| Title of Invention | "A PROCESS FOR THE PREPARATION OF RECOMBINANT GROWTH HORMONES FROM DIFFERENT ANIMAL SPECIES WITH DAIRY AND VETERINARY IMPORTANCE" |
|---|---|
| Abstract | The process of the present invention for the preparation of recombinant animal somatotropins ( growth hormones) from selected animal species viz. goat, buffalo, Indian exotic cattle essentially comprises of preparing a complementary DNA (cDNA) encoding for the mature hormone polypeptides employing either chemical synthesis, or by the methods of recombinant DNA , or a judicious combination of both. A preferred method involves the reverse transcription by RNA-dependent DNA polymerase enzyme, followed by selective amplification of the cDNA species encoding for the growth hormone using thermostable DNA-dependent DNA polymerase, specific oligonucleotides. The genetic elements encoding for the growth hormones are then transferred into heterologous hosts selected from bacteria, yeasts, animal cells with a recombinant vehicle carrying hyper expressive growth hormone cDNA and expressed into polypeptides. These polypeptides are then further recovered in their biologically active forms from the heterologous hosts in highly purified state by the process of the present invention. |
| Full Text | This invention relates to a process for the preparation of recombinant hormones of different animal species with dairy and veterinary importance. More particularly , this process relates to animals like the Indian species of cattle, water buffalo, goat, camel, donkey. In India the cow has been revered as sacred animal since time immemorial. This stems from the many-splendor offering rendered by the cow to mankind in the form of milk. Indians have held milk in high esteem through the ages , considering it as food par excellence. This attitude , inherited for centuries , has made India the largest consumer market for milk and milk-products. It provides some 95 per cent of animal proteins and almost 100 per cent of animal fat in the daily diet of Indians. Today , milk is the country's number one farm produce in terms of its contribution to the national economy [ A.C.Kulsherestha , Contribution of livestock to national income. Pp:77-80 , In dairy India , 1997 (fifth edition ) Ed. By P.R. Gupta A-25 Priyadarshani Vihar Delhi -110092]. Presently , some 70 million , Indian farm families are maintaining a gigantic milch herd of nearly 100 million , significantly constituted of cows. However , no less important than the cow is the buffalo , which is considered India's milk machine , constituting nearly 40 percent of the bovine population and contributing more than half ( 52 per cent ) of the total milk production in the country. India has more than 50 per cent of the world's buffalo population with high yielding breeds, like the 'Murrah' ( aptly called the Holstein -Friesian of the buffalo world) [ M Sasaki ( FAO, Bankok), Asian buffalo: small farmer's asset. Pp :119-121 , in Dairy India , 1997 ( fifth edition) Ed. P.R. Gupta ]. It is reputed for its feed conversion efficiency , converting low-grade fibrous feed into high value milk , with higher (i.e. higher than cow's milk) total solids ( 33 per cent ) and fat ( 7-15 per cent) content. The goat, apart from being the most important meat animal in India , contributes nearly 3 per cent ( around 2 million tones ) of total milk produced in the country. The goat is considered to be an asset particularly for the small and marginal farmer alike [ S. Krishnamurthy , Goat milk production : an unknown element. pp : 122-124 , In Dairy India 1997 ( fifth edition) Ed. P.R. Gupta ] . India , with its large reserve of goats ( e.g. Black Bengal) are renowned worldwide for quality and taste of their meat. However , these animals suffer from limitation of poor growth rate. Although cattle slaughter is not allowed in most parts of the country due to religious reasons, the buffalo has the potential to be a good meat animal. To procure 75 million metric tonnes of milk, India maintains a huge population of milch animals of nearly 60 million cows and 40 million buffaloes. This is basically due to the poor productivity by the indian non-descricptive native breeds of animals, which is further aggravated by poor feeding, health-care and management. Against the world's average milk production of around 2000 kg or more per lactation and the highest, of nearly 9000 kg per lactation (in Israel), the average yield in India is only 987 kg [R.P. Aneja and B.P.S. Pun. India's dairy riddle unravelled. Pp: 4-26. In Dairy India, 1997 (fifth edition) Ed. By P.R. Gupta], In order to make milk available at a lower, affordable price, therefore, there exists a compelling need for improving the productivity of milch animals. Apart from better feeding and management, improvement of the genetic merit of indigenous cattle by traditional selection, the use of superior germplasm of exotic breeds (cross-breeding) through artificial insemination etc., can also potentially result in a remarkable enhancement of milk production in the country. This technology, however has its own peculiar limitations. For example, incorporating more than 50 per cent exotic germplasm was found counterproductive. This is basically due to reduced adaptability to harsh temperate climatic conditions prevailing in the countrv and the inability to sustain poor feeding and management conditions by the cross-bred animals. All this culminated into increased disease susceptibility, higher rates of infertility and. finally, an overall poor productivity. It was known as early as the 1930's that the injection of bovine pituitary extracts into dairy cows increases milk yield (Evans, H.M. and Simpson, M.E., 1931., Am. J. Physiol, 98: 5111931; Asimov, G.J. and Krouze, N.K.. 1937.. J. Dairy Sci.,USA. 20: 289). This important endocrine factor was later identified as growth hormone (bGH), also called somatotropin (Li. C.H., M.H. Evans and M.E. Simpson. 1945.. J. Biol. Chem.. 159: 359). However, the limited supply and impurity of pituitary-derived bGH precluded its early commercial exploitation. With the emergence of biotechnology in the 1980's it became possible to produce bGH in large quantities through recombinant DNA processes. Subsequently, many pharmaceutical companies viz. Monsanto. Elanco. Upjohn, American Cyanamid etc. have developed recombinant bGH (rbGH) and its derivatives in the United States and Europe. However, all these products essentially pertain to the species of cattle native to these countries: the cattle indigenous to the indian sub-continent is considered sufficiently different from the 'western' variety (Bos taunts) to be considered a separate species (Bos indicns). Hence, to optimally boost the dairy output from this variety, its intrinsic growth hormone needs to be available. Likewise, growth hormones specifically for animals of veterinary and dairy importance for other animals of the sub-continent and adjoining geographical areas of India are still largely unavailable. It has now been firmly established that with the use of rbGH, milk yield increases in the range of 10-25 per cent in exotic cattle (see, in this context: Bauman, D.E., Eppard. P.J.. DeGeeter, M.J. and Lanza. G.M.. 1985., J. Dairy Sci., USA, 68: 1352; Peel. C.J. and Bauman. D.E. 1987., J. Dairy Sci., USA. 70: 474; Hodate. K.. Ozawa, A. and Johke, T., 1991.. Endocrinol. Jpn.. Japan. 38: 527; Armstrong. J.P.. Harvey. R.W.. Poore. M.A., Simpson, R.D.. Miller. D.C.. Gregory. G.M. and Hartnell. G.F.. 1995. J. Anim. Sci. 70: 3051-3061) and upto 30 per cent in buffalo (Ludhri, R.S., Upadhyay, R.C., Singh, M., Gunarante, J.R.M. and Basson, R.P.. 1989., J. Dairy Sci., USA, 7: 2283; Ludri. R.S.. Upadhyay. R.C.. Singh. M.. Singh. C.B.. Dhaka. J.P.. Chandrashekhar. T.. Nair. S.. Tomer. O.S., Ghosh. M.K., Verma. G.S. and Singh. O.. 1997 Annual Report. NDRI. Karnal). without added cost on feeding, essentially due to improved feed-conversion efficiency. It is worth mentioning here that an increase of 20 percent milk corresponded roughly to 10 per cent lower feed cost per kg milk produced (Gravert, H.O., 1988.. IDF Document, 72nd Annual Session of the IDF. Budapest). Exogenous administration of rGH has also been found to improve growth rate, feed conversion efficiency and carcass composition in cattle, sheep and pig (reviewed by Enright, W.J.. 1989., and Hanrahan. T.J.. 1989.. In 'Use of Somatotropin in Livestock Production'. Elsevier Science Publishers Ltd., England, pp: 132 and 157; Myers. M.J., Farell. D.E.. Evock-Clover. C.M., Cope, C.V., Henderson, M. and Steele, N.C.. 1995., Pathobiology. Switzerland. 63: 283; Evock-Clover. C.M., Mayer. M.J. and Steele. N.C.. 1997 J. Anim. Sci.. USA. 75: 1784). satisfying the commercial requirement of an effective carcass enhancer. Thus, perhaps no other single technology has shown as much potential to revolutionize animal productivity as GH. It remains a fact that with the population explosion in India, nearly 1000 million people are in stiff competition with its vast livestock animal population for every piece of grain and land. A large proportion of these animals freely graze on agricultural fields and forest areas posing serious threat due to degradation and denudation of land and loss of natural resource. The huge quantity of methane gas produced by these animals (methane gas is produced in ruminant animals due to microbial digestion of feedstuff causing damage to the ozone layer) has also become an important issue of growing concern by the world communities. In this context, the importance of rGH is not only to boost the overall productivity of the existing herd but more importantly it enables one to produce the same amount of milk or meat from less number of animals. However, the animal species important to the indian sub-continent/south asian region are unique and quite distinct from those found in the western world where the technology for recombinant GH has been developed. Apart from the fact that the species of cattle in the indian sub-continent (Bos indicus) is different from that in the western countries (Bos taunts), carrying with it the finite possibility that the rGH developed earlier in the west is distinct from that intrinsic to the indigeneous cattle population, the growth hormones of some of the other genera of animals of veterinary importance in the indian subcontinent viz. water buffalo (Bitbalns bnbalis) and Indian Beetal goat (Capra hircus) have not been hitherto produced by rDNA technology even though these are evolutionarily far removed from the bovine hormones. The role of growth hormones in augmenting animal productivity is now well-established. This protein hormone is produced in the pituitary gland of all animals, and is an essential endocrine factor for the physiological growth and lactation in mammals (Lewis, U.J. 1992.. Trends Endocrinol. Metab. 3: 117). The mammalian GH is a single-chain polypeptide of about 190 amino acids with a molecular mass of 22,000 daltons. produced and secreted by a specialised subset of cells of the anterior pituitary gland under the influence of the hypothalamic factor, viz. growth hormone releasing factor (GRF). The growth promoting ability of GH was known as early as the 1930s (Evans. H.M. and Simpson. M.E., 1931., Am. J. Physiol, 98: 5111931; Lee, M.O. and Schaffer. N.K.. 1934., J. Nutr. 7: 377). Later on it was also found to possess galactopoetic (enhancer of milk synthesis) properties (Asimov. G.J. and Krouze. N.K., 1937.. J. Dairy Sci.USA. 20: 289; Young. F.G., 1947.. Brit. Med. Bull. 5: 155). Since then, efforts were on towards exploiting these two vital properties of GH for human health and animal productivity purposes. But the studies as well as the applications were seriously restrained by the limited availability and impurities of the hormone preparation obtained from animal cadavers, until the emergence of recombinant DNA technology in the 1970s. With the advent of the gene cloning techniques sufficient quantities of highly purified GH from a number of species have ibeen produced. Thus, the main object of the present invention is to provide a process for the preparation of somatotropin proteins of selected animal species by recombinant DNA methodology followed by conventional protein purification to obtain biologically active hormone(s) in highly pure form. Another object of the present invention is to provide a process for preparing highly pure and biologically active somatotropin monomers from somatotropin dimers and higher oligomers produced by the host organism in which the genes encoding these polypeptides are expressing using recombinant DNA methodology. Yet another object of this invention is to provide a method for the isolation and recovery of somatotropin monomers from solutions containing somatotropin monomers and oligomers in the absence of residual host cell proteins and other contaminants. The process of the present invention for the preparation of recombinant animal somatotropins from selected animal species viz. goat, buffalo, Indian exotic cattle etc, essentially comprises of preparing a complementary DNA (cDNA) encoding for the mature hormone polypeptides employing either chemical synthesis, or by the methods of recombinant DNA research, or a judicious combination of both. A preferred method involves the reverse transcription, by RNA-dependent DNA polymerase enzyme, of the messenger ribonucleic acid (mRNA) fraction prepared from the pituitary glands of the animals, followed by selective amplification of the cDNA species encoding for the growth hormone using thermostable DNA-dependent DNA polymerase, specific oligonucleotides etc and subjecting the aforesaid reaction to several cycles of thermal cycling by the well-known polymerase chain reaction )PCR) technique. The genetic elements encoding for the different growth hormones are then transferred into heterologous hosts such as bacteria, yeasts, animal cells etc and expressed into polypeptides. These polypepties are then further recovered in their biologically active forms from the heterologous hosts in highly purified state by the process of the present invention. Accordingly, the present invention provides a process for the preparation of recombinant growth hormones of important animals species with dairy and veterinary importance, useful for the enhancement of metabolic processes such as enhanced lactation; said process characterized in that recombinant growth hormone being obtained by downstream splicing of growth hormone cDNA into a synthetic hyper expressive cistron synthesized from a two-cistronic expression systems no. BGHB1 and BGHB2 as described herein, and incorporating the entire two-cistronic cassette into a vehicle as describe herein to get hyper expressive growth hormone , the said process comprises: (i) synthesis of the complementary DNA (cDNA) encoding for the growth hormone proteins of various animal species of veterinary importance, such as the Indian water buffalo, Indian species of cattle (cow), goat, camel, donkey , yak either by chemical synthesis such as herein described , or by biochemical means using appropriate nucleic acid synthesizing enzymes as described herein , or a combination of these two methods. (ii) recombining the growth hormone-encoding cDNA so obtained with a vehicle by downstream splicing of GH cDNA to a synthetic cistron as described above and cloning the vehicle in a heterologous host cell selected from a virus, bacterium, yeast, animal or plant cell to get recombinant vehicle carrying hyper expressive GH cDNA, (iii) introducing the recombined vehicle so obtained into an appropriate heterologous host as defined above by conventional methods as described herein to get transformed cells, (iv) isolating transformed cells carrying the hyper expressive growth hormone cDNA by conventional manner, (v) expressing the growth hormones in the transformed host cells of step (iv) in an appropriate medium as described herein using conventional methods as described herein , (vi) isolating the polypeptides of the growth hormones from the culture of the host cells and subjecting them to various fractionation and chromatographic methods as described herein to obtain highly pure desired growth hormones. The details of the process of the present invention begin with preparing a polymeric deoxynucleic acid (DNA) molecule encoding for the mature growth hormone polypeptides of the respective species. This step was accomplished through known methods of synthesizing nucleic acids whereby the sequence of the polymeric DMA corresponds to the sequence of nucleotides encoding the growth hormone polypeptides in the pituitary gland of the particular animal. The pituitary gland can be also used as a source for extracting the ribonucleic acid (RNA) component from the tissue by well-known procedures. This preparation of RNA is then utilized to prepare a complementary DNA (cDNA) encoding for the mature hormone polypeptides by the methods of recombinant DNA research (References: Ausubel, F.M., Brent, R., Kingston. R.E.. Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, k., 1987., 'Current Potocols in Molecular Biology', Green Publishing Associates and Wiley-lnterscience, New York: Sambrook., J., Fritsch. E.F. and Maniatis, T., 1989., " Molecular cloning , a laboratory manual", Cold Spring Harbor Laboratory Press, New York). A preferred method involved the reverse transcription, by RNA- dependent DNA polymerase enzyme, of the messenger ribonucleic acid (mRNA) fraction prepared from the pituitary glands of the animals, followed by selective amplification of the cDNA species encoding for the growth hormone using thermostable DNA-dependent DNA polymerase, specific oligonucleotides etc and subjecting the aforesaid reaction to several cycles of thermal cycling by the well-known polymerase chain reaction (PCR) technique. The DNA containing the genetic elements encoding for the different growth hormones are then transferred into heterologous hosts after linking them with a suitable cloning vehicle and expressed into polypeptides. These growth hormone polypeptides were then recovered in their biologically active forms from the heterologous hosts in highly purified state by the process of the present invention. Thus, by virtue of the present process, the growth hormones were produced in a much enhanced level in the recombinant host as compared to their natural source and these were then recovered in a highly pure state in a biologically active form so as to be utilised for various useful applications. In an embodiment, the invention provides a method for the preparation of biologically active growth hormones in pure form from selected animals of veterinary importance indigenous to the south asian and Indian sub-continent region/s. namely indian water buffalo, indian exotic cattle species, goat, camel, donkey, yak and the like. In another preferred embodiment of the process of the present invention, the cDNA encoding for the mature forms of the growth hormones is prepared by using specific enzymes viz.. DNA-dependent RNA polymerase that cam- out reverse transcription of the messenger RNA (mRNA) isolated from the pituitary glands of the animals whose gro\\th hormones are to be prepared. In another embodiment, the GH encoding cDNAs are ligated with a plasmid vehicle capable of either autonomous replication or after integration into the chromosomal DNA in a suitable host cell, such as bacteria, yeast and the like. In yet another embodiment, bacterial promoters such as the tac promoter. T7 RNA polymerase promoter and the like are utilised for the hyper-expression of growth hormone genes in a heterologous host cell such as E. coli. In another embodiment, the growth hormone polypeptides are produced intracellularly in a host cell such as E. coli. In another embodiment, the growth hormone polypeptides are produced intracellularly in a host cell such as E. co/i in the form of insoluble refractory- bodies (inclusion bodies; IBs) that can be isolated after lysis of the host cell and dissolution of the IBs in solutions containing chaotropic agents, such as urea, guanidine hydroohloride and the like. In another embodiment, the growth hormone polypeptides isolated from" the IBs are allowed to refold to their biologically active conformations concomitantly reforming their native disulflde pairings from the reduced state by air-oxidation. In another embodiment, the growth hormone polypeptides isolated from the IBs are allowed to refold to their biologically active conformations and to reform their native disulflde pairings from the reduced state through catalysis by a mixture of reduced and oxidised glutathione. In yet another embodiment, the growth hormones obtained by the dissolution of IBs and then oxidatively refolded are isolated after the completion of the reoxidation/rcfolding reaction by contacting and binding with a suitable insoluble chromatrophy matrix, such as one containing charged groups, specific or non-specific affinity ligands. or ones containing functional groups/ ligands suitable for hydrophobic interaction with proteins such as methyl-, butyl-, hexyl-. phenyl-. decyl- and the like with the growth hormone so as to obtain the biologically active, refolded grouth hormones devoid of the chaotropic agents in the refolding mix. In another preferred embodiment, the matrix utilised for isolating the growth hormone from the refolding reaction contains decyl groups. The invention and its embodiments are illustrated by the following examples, which, however, should not be deemed to limit the scope of the invention in any manner. Various modifications that may be apparent to those skilled in the art are deemed to fall within the scope of the invention. Advantages of the present invention By the process of the present invention, the biologically active forms of the growth hormones of several unique animal species of Indian origin viz., indian species of cattle (Bus indicus) which is phylo-genetically distinct from the taurine cattle (Bos taunts), indian water buffalo (Biibalus bnbalis), Beetal goat (Capra hircus) etc, that are of tremendous economic importance, can be prepared by employing recombinant DNA methodology. The nucleic acid sequence of protein coding region of GH cDNAs from Indian species of cattle, buffalo and goat were found different from that of already reported sequences of related animals viz. taurine cattle and sheep. The indian water Buffalo is an unique species in the bovidae family predominantly found in this subcontinent. The indian variety of goat (Capra hircus: vem. 'Beetal goat1) is the most important meat animal of the country and its growth hormone possesses a sequence distinct from all the known growth hormones, as mentioned above. .Similarly, the growth hormone of indian species of cattle (Bos indicns). which is considered to be a bovine species distinct from the taurine cattle of the western world (Bos taunts), can be prepared in large amounts through the process of the present invention, as can also the hormones from the goat and buffalo. From a biotechnological viewpoint (as in stimulation of milk or meat production with GH). it may be advantageous to use the homologus GH in the respective species since unwanted side-effects, including possible immune reactions, associated with the relatively long-term introduction of a growth hormone in a heterologous species of animal are likely to be generated. Thus, there has been an acute need to be able to produce larger quantities of recombinant hormones from species of economically important animals that are predominantly found in the indian subcontinent/South Asia. The process disclosed in the present invention for the production of recombinant grouth hormones addresses this vital need. The process of the present invention also utilizes a much rapid and simpler method of isolation of the respective growth hormone-encoding genetic determinants. This is based on a rapid single-step RNA isolation method which effects a targeted amplification of GH-mRNA through RT-PCR technique, generating a single species of enriched clonable cDNA molecules, thus eliminating the need for constructing a cDNA library and a process of screening for the desired clone. Additionally, one of the preferred "host-vector" systems used for the production of these recombinant polypeptides produces the native (alanyl) form of the hormones without any non-native N-terminal extension. The expression system efficiently removes the N-terminal formyl-Methiomne from the GH polypeptides in the host-cell as no traces of it were noticed. Brief Description of the Figures Figure 1 Agarose gel electrophoresis of RT-PCR amplified GH-cDNA derived from the pituitary gland of Bos indlcus. The preparation of crude RNA extracted from the pituitary glands of the different animal species were subjected to RT-PCR reaction using a pair of taurine cattle GH-cDNA sequence specific primers. Approx. one-tenth (10 µl of a total volume of 100µl of the amplification reaction) product was resolved through agarose gel (0.8%) electrophoresis, stained with EtBr, and photographed against transmitted UV light. Lane 1: 100 bp DNA ladder: lane 2: PCR reaction control, showing amplification of about 0.8 kb control DNA template: lane 3: RT-PCR control, showing amplification of a 1.2 kb mRNA template; lane 4: RT-PCR amplification product (approx. 580 bp) of cattle pituitary RNA preparation, and lane 5: Hindlll and EcoRI digested λ-DNA markers. Figure 2 Agarose gel electrophoresis of RT-PCR amplified GH-cDNAs from pituitary gland of Indian water buffalo (Bubalus bubalis) and Indian beetal goat (Capra hircus) . Either total RNA or purified poly-A+ RNA preparations from the pituitary glands of the different animal species were subjected to RT-PCR reaction using a pair of taurine cattle GH-cDNA sequence-specific primers (see section: General Methods Used in Examples, for details). Approximately one-tenth of the amplified reaction product was resolved through agarose gel (0.8% w/v) electrophoresis. stained with EtBr. and photographed against transmitted UV light . Panel A Lane 1: Hindlll digested λ-DNA marker: lane 2: RT-PCR product (double stranded cDNA) resulted using random primers, and lane 3: RT-PCR product generated using oligo-dT primer, during the first strand synthesis reaction step. Panel B Lane 1: Hindlll digested λ-DNA marker; lane 2: RT-PCR product resulted using Pfu DNA polymerase. and lane 3: RT-PCR product generated using Taq DNA polymerase: lane 4: 'negative' PCR control reaction (i.e.. carried out without any added template), and lane 5: Hindlll digested λ-DNA markers. Figure 3 A, B Nucleotide sequences and the comparative alignment of different GH cDNAs. The complete nucleotide sequence of protein-coding region of GH cDNAs for Indian species of cattle (Bos indicus), Indian water buffalo (Bubalus bubalis) and Indian beetal goat (capra hircus) were determined as detailed in 'General Methods used in Examples', and then aligned with that of exoti; cattle (Bos taunts: Miller. W.L.. Martial, J.A and Baxter. J.D.. 1980.. J. Biol. Chem. 255: 7521) and sheep (Warwick, J.M.. Wallis. O.C. and Wallis. M.. 1989.. Biochim. Biophys. Acta. 1008: 247). The regions of differences in the sequences are boxed. (BT = exotic taurine cattle: BI = Indian cattle: BB = Indian water buffalo: GB = Indian beetal goat, and SH = sheep). Figure 4 Nucleotide sequence corresponding to the N-terminal region of the GH-open reading frame (GH-ORF) in the expression vectors pUGH99A and pUG99II/B. The sequencing reactions were carried out using a GH sequence specific primer (UMI), the 3'-end of which is located about 100 base away from the 'A' of the start codon. ATG as detailed under the section 'General Methods used in Examples'. The portions of autoradiograph of 6% urea-acrylamide sequencing gels showing : (a) the important translational elements (Shine-DclgaiuG. SD, and initiator codon. ATG) and perfectly incorporated first ainino acid of GH (Ala) through a synthetic Ncol-linker in pUGH99A (the initial expression clone where native GH cDNA sequence was placed directly under trc promoter and resulted GH expression albeit at a low level): (b) the sequence of the entire synthetic cistron in pUG99II/B (the hyper-expressing clone for GH: see Examples for details) with ribosome binding sites of both cistron is indicated. Figure 5 Level of intracellular accumulation of recombinant growth hormones (rGH) by various E. coli strains. The strains containing GH expression plasmid, pUG99IIB. were grown to an OD600 of approx. 0.3 unit and induced with ImM (final concentration) of IPTG. The pre- and post-induced cells (after different hours of growth) were harvested, lysed and resolved on 12.5% SDS-PAGE. An equal number of cells (equivalent to 20 µ1 culture of one OD600 unit) corresponding to the bacterial growth at different hours for all the E. coli strains (A= XL 1-Blue: B= BL-21; C= Toppl: D= Topp2 and E= Topp3) were analysed (see: General Methods Used in Examples). Across all the gels: lanes 2-8, represent, respectively, 0. 0.5, 1, 2, 4, 8 and 16 h of post-induced lysates, whereas lane 1 represents control lysate containing 5µg of std. pituitary bGH. and lane 9 shows standard molecular weight markers [with the bands from top to bottom of the gel as follows. 97.4 kD (phosphorylase b). 66.2 kD (albumin). 42.7 kD (ovalbumin). 31 kD (carbonic anhydrase), 22 kD (bovine growth hormone = 2.5 µg), 21.5 kD (soy bean trypsin inhibitor) and 14.4 kD (a -lactalbumm)]. Essentially similar results were obtained with the expression studies with the recombinant GHs of the other two animal species. Figure 6 SDS-PAGE analysis of rGH-containing inclusion bodies. Panel A: Shows the sedimentation of goat rGH-IBs at different "g" values where the crude lysate (lane 1) and pellets of 100 (lanel), 200 (Tone 4), 1000 (lane 6). 2500 (lane 9). 5000 (lane 12) and 10000 (lane 15) x g's are shown. Lanes 3 and 5 are the supematants of 100 and 200 g's respectively, whereas lanes 7, 10, 13 and 16 show pellets, and lanes 8, 11, 14 and 17 show supematants of high speed resedimentation (10,000 g) of supernatants of 1000, 2500, 5000 and 10000 x g's. respectively. Panel B: shows the effect of washing of GH-IBs with different concentrations of urea on quality of rGH obtained. Lanes 1, 3, 5 and 7 are the pellets and lanes 2, 4, 6 and 8 are supematants of 4, 5, 6 and 7 M urea washing respectively. Panel C: shows washing of GH-IBs with urea-Triton X-100 combinations. Lanes 1, 3 and 5 represent pellets and lanes 2, 4 and 6 represent supernatants of 3. 4 and 5 M urea with 0.5% Triton X-100 washing respectively. Closely similar results were obtained in case of rGH from cow (Box indicus) and buffalo (Bubalus bnbalis). Figure 7 Reverse-Phase HPLC analysis of the reduced and oxidised forms of recombinant Growth Hormones. Approximately 25 µg of either oxidised sample or unreduced (native) pituitary bovine growth hormone. bGH (in 10 mM EDTA) was used for analysis. Reduced samples were prepared by reducing the pit. bGH or oxidised/refolded rGH sample with 20 mM (final cone.) of dithiothreitol (DTT) at room temperature for 30 min immediately before HPLC analysis (see section: General Methods Used in Examples). A C-4 reverse phase HPLC column was used for these separations. A step-gradient was applied over a period of 50 min, as detailed under General Methods Used in Examples, and the column was run at a flow rate of 300 µl/min. The absorbance was continuously recorded at 214 nm and data were analysed by Applied Biosystem's 600 Data Analysis System. A. Unreduced std. pit. bGH (native) B. Reduced std. pit. bGH C. Sample retrieved at 0 h of air-oxidation of rGH D. Sample retrieved after 3 h of air-oxidation of rGH E. Sample retrieved after 36 h of air-oxidation of rGH F. - Reduced sample E (36 h of air-oxidised rGH) G. Unreduced HIC-purified rGH. Figure 8 Oxidation kinetics of recombinant goat growth hormone (rgGH) showing relative abundance of oxidised and reduced forms. The various rGH inclusion bodies were dissolved in 8 M GdCl and subsequently oxidative refolding in 6M GdCl was allowed to take place at a final protein concentration of 1.5 mg/ml in an open container at room temperature for a total duration of 72 h, as detailed under the 'General Methods Used in Examples' section and Examples 8 and 9). Aliquots were removed periodically and the oxidation reaction stopped by the addition of EDTA to a final cone, of 10 mM. Approximately 25 µg of oxidised protein sample was resolved through RP-HPLC. The relative area (per cent of total) under oxidised monomeric (open circles) and reduced (closed circles) peaks at different times post-oxidation, obtained by integration with a computer, and are plotted. Samples were also analysed for free thiol content. The estimated free thiol (-SH) content at different times during the refolding reaction is expressed as µmol -SH groups per µmol of protein sample. Figure 9 Purification of oxidised rgGH by hydrophobic interaction chromatography (HIC). The refolded protein (rgGH) bound to the HIC matrix (Decyl-agarose: see Example 10, and section 'General Methods used in Examples') was eluted successively with 50 ml each of 50 mM Tris.HCl. water, and 8 M urea solution Fractions (each approx. 5 ml) were analysed for protein content. The absorbance (at 595 nm) of water and urea-eluted fractions are plotted. The vvater-eluted peak was found to contain contaminating proteins but no GH. whereas the peak eluting with 8 M urea contained highly enriched rgGH. as observed by SDS-PAGE analyses. Very similar results were obtained in case of the oxidation of the recombinant GHs of buffalo and indian cattle species. Figure 10 Purification of monomeric rgGH from HIC-purified sample by ion-exchange chromatography (IEC). The protein obtained from HIC was pooled as described in Example 10 and section 'General Methods used in Examples' and loaded onto a DEAE-Sepharose containing column equilibrated with 5 mM ammonium bicarbonate buffer. Bound protein was eluted with a linear gradient (5-. 1000 mM) of ammonium bicarbonate. Fractions of approximately 4 ml were collected and the protein content was estimated in each. An identical 'blank' gradient was also run for the correction of baseline, as ammonium bicarbonate in presence of Bradford's reagent was found to contribute significantly to the absorbance. The corresponding blank values were subtracted from the absorbance values of each fraction before plotting (for details, see section 'General Methods Used in Examples'). The major peak eluting at around 400 mM salt representing highly purified monomeric rgGH. Very similar results were obtained in case of the purification of the recombinant GHs of buffalo and indian cattle species by IEC. Figure 11 Non-reducing SDS-PAGE analysis of rGH to establish monomeric nature of the correctly folded GH proteins. The oxidation reaction of goat rGH was stopped by adding 10 mM EDTA (final concentration) into the samples retrieved at different durations of air-oxidation. To remove GdCl. oxidised samples were TCA precipitated and the resultant pellets were washed with 20% acetone (containing 10 mM EDTA). The unreduced pit. bGH (taken as oxidised control of GH) was also processed similarly. The reduced samples of pit. bGH as well as rGH were prepared by incubating the protein in the presence of 20 mM (final concentration) DTT at room temperature for 30 min. following which the reduced samples were also subjected to TCA precipitation and acetone washing. The air-dried pellets were finally dissolved in 1 x modified SDS-PAGE buffer (MSB) without reducing agent. A part of these samples were resolved through 12.5% SDS-polyacrylamide gel containing 10 mM EDTA (see 'General Methods used in Examples' for details). Panel A: Lane 1: oxidised (unreduced) pituitary bGH standard; lane 2: 0 h. lane 3: 3 h and lone 4: 36 h of oxidised r GH: lanes 5 and 6 are unreduced HIC and DEAE purified materials, respectively: and lane 8: reduced pituitary' bGH standard. Panel B: Lanes 1 (2.5 µg) and 2 (7.5µ.g) showing unreduced std. bGH and lanes 3 (lµg) and 4 (5µg) showing unreduced DEAE-agarose purified rGH. Same samples (std. pituitary bGH and rGH) in exactly similar quantities and sequences (as above) were am between lanes 6-9 after reduction. Virtually identical results were observed in case of the refolded and purified recombinant GHs from buffalo and indian cattle species. Figure 12 SDS-PAGE analysis of recombinant goat Growth Hormone at different purification stages. The IPTG-induced recombinant E. coli cells (Topp2 strain, harbouring GH hyper-expression plasmid pUG99II/B) were centrifuged and then lysed. The rGH IBs were harvested from crude lysates by centrifugation and the IB-pellets were washed with urea-Triton X-100. The purified GH-IBs were subsequently dissolved in 6 M GdCl and air-oxidised. The oxidised rGH was then purified by a two-step chromatographic method. In the first step, oxidised rGH was purified through the hydrophobic interaction matrix (Decyl-agarose). The HlC-purified rGH (eluted in 8 M urea) was then further purified by DEAE-Sepharose chromatography. Aliquots of rGH samples collected at individual purification steps were analysed on a 12.5% SDS-polyacrylamide gel. Lane 1 (MW) showing, molecular weight standards with indicated mass: lane 2: crude lysate; lane 3: purified IBs; lanes 4-7: HlC-purified (respectively, 5, 10, 20 and 60 µg) rGH: lanes 8-11: DHAE-punticd (respectively, x 10. 20 and 40 µg) rGH and lane 12: std. pit. bGH (10 MS)- Figure 13 A, B. N-terminal amino acid sequence of purified recombinant growth hormone proteins. Approximately 20µg each of PVDF membrane (electro)-blorted samples were sequenced by gas phase Edman's sequential degradation method on an ABI's 476A protein sequencer system (see 'General Methods used in Examples' for details). The HPLC profiles of amino acid phenylthiohydanation derivatives of standard PTH-amino acid mixture, and first ten cycles of sequencing of recombinant rGHs are shown. The relevant amino acid peaks in each cycle are encircled. The peak at around 6.5 min across all the cycles represents dimethyldiphenylthiourea (dmptu). Panel A: the HPLC profile of amino acid phenylthiohydanatoin derivatives of standard mixture and first ten cycles of gas phase sequencing of recombinant buffalo GH. Panel B. the HPLC profile of amino acid phenylthiohydanation derivatives of standard mixture and first ten cycles of gas phase sequencing of recombinant goat GH. Figure 14 Radio-receptor binding assay for biological activity of refolded and purified recombinant growth hormones. In a competitive assay, different amounts of either unlabelled standard N.I.H. pituitary bovine GH (closed circles), buffalo rGH (triangles) and goat rGH (solid squres) were allowed to bind to goat GH-receptor preparations (equivalent to approx. 150 µg of protein) in the presence of 125I-labelled pituitary bGH (containing approx. 10.000 CPM). The specific binding (values shown are the means of two independant determinations) as CPM are plotted against ng of competing protein. Figure 15 Expression construct: Developed for the hyper-expression of GHs Synthetic two-cistronic GH-expression systems were constructed under trc promoters in pTrc99A. In this system GH cDNAs of all the three different species were used as second cistron and placed under a short synthetic first cistron. The entire expression cassette is localised between the EcoRI and BamHI sites in the vector. Various translational signals viz.. SD. ATG, stop codon etc are indicated (hiked and boxed) General Methods Used in Examples In general, the methods and techniques of rDNATwell known in the area of molecular biology. These include plasmid DNA isolation and characterization, restriction endonuclease digestions, isolation of DNA fragments. PCR methods, preparation of synthetic oligonucleotides. dephosphorylation and kinasing of DNA. DNA-DNA ligations. transformation of different hosts with rDNA molecules etc, as well as procedures of protein biochemistry, such as SDS-PAGE electrophoresis. column chromatography of various kinds etc that are well known to practitioners of modern biology that are skilled in the art. These procedures are readily available in current scientific literature as well as from several standard protocol treatises and manuals pertaining to this field (for example. Ausubel. F. M. Brent. R.. Kingston, R. E., Moore. D. D.. Seidman, J. G.. Smith. J. A. and Struhl, K.. 1987.. 'Current Protocols in Molecular Biology'. Green Publishing Associates and Wiley-Interscience, New York Sambrook. J., Fritsch, E.F. and Maniatis, T., 1989., "Molecular cloning, a laboratory manual". Cold Spring Harbor Laboratory Press, New York; 'Guide to protein purification' (M.P. Deutscher. Ed.). Methods in Enzymology, vol. 182, Academic Press. New York: J.C. Jansen and L. Ryden. 1989., 'Protein purification: principles. high resolution methods, and applications., VCH Publishers, New York). However, unique methods or protocols, if used, with relevant details in the context of specific experiments, only are described herein, wherever relevant. Growth, induction, harvesting and lysis of cells for analysis of level of expression Inoculum was raised by seeding 5 ml LB-Ampicillin (100µ,g/ml) with a single, well-isolated colony (approximately 2 mm in diameter) from a freshly grown plate (plate streaked from -70°C glycerol stock about 12-16 h before use) of relevant culture and grown overnight (12-16 h) in a/shaker-incubator (37°C. 200 rpm). Next day, 5-10 ml of medium with the appropriate antibiotic was seeded with overnight grown culture at 0.1% (v/v) level and grown upto an OD600 of about 0.3. For comparative studies on the levels of expression obtained by different E. coli cultures, the OD600 of overnight cultures were also checked with 10-fold dilution (for more accurate estimation) and. accordingly, an equivalent amount of seed culture was used for inoculation. At 0.3 OD600 an appropriate volume of culture was induced with 1 mM (final concentration) IPTG and grown for upto 16 h depending upon the requirement. Just before induction, 1 ml aliquot of pre-induced culture was taken out and the pellet (obtained by centrifllgation at 14,000 rpm for 30 sec at room temperature in an Eppendorf microcentrifuge) was preserved at -70°C. After the end of intended time-period of growth. 1 ml aliquots of post-induction cultures were taken out and harvested as above. Cell pellets were resuspended in 50-100 u.1 of distilled water and lysed by adding 50-100 ul of 2x modified sample buffer (MSB) [1 x MSB: Tris HC1 of pH 6.8 (150 mM). SDS (2%, w/v). Glycerol (15%, v/v), BME (7.5%, v/v) or DTT (100 mM), BPB (0.01%. w/v) and Urea (3 M)]. Raising of anti-GH antibodies To raise anti-GH polyclonal antisera, 100 µg of standard pituitary bGH (obtained from Dr. A. F. Parlow, Director, Pituitary Hormone and Antisera Centre, Harbor-UCLA Medical Center. Torrance. California 90509) was dissolved in a final volume of 1 ml in PBS. to which 1 ml of Freund's complete adjuvant was added. The emulsified mixture of antigen and adjuvant was injected subcutaneously at four different sites (0.5 ml/site) into an outbred (New Zealand white) rabbit. Four boosters of 50 µg antigen each were also given every 20 days starting from 4 weeks after primary immunization. Animals were bled 7-10 days after every booster immunization, for testing of antibody titre. At the end of the immunization schedule serum was separated and stored at -70°C in 1 ml aliquots. Also, pre-immune sera were prepared before the beginning of the immunization regimen and was stored similarly till used. Immunodetection of Western blotted proteins The resolved proteins on SDS-PAGE were electrophoretically transferred onto NC-membrane (Towbin. H. and Gordon. J.. 1984.. Immunol. Methods 72: 313). Non-specific binding sites on protein-blotted NC-membrane was blocked by dipping the membrane in a solution of 3-5% skim milk powder in PBS (pH 7.4), either for 2-3 h at room temperature or overnight at 4°C. The membrane was then incubated with 1:1.000 dilution of anti-GHrabbit antiserum in blocking solution for 1-2 h at room temperature with gentle shaking. Then the membrane was washed with PBS (3-4 changes of 100 ml buffer for 15 min each). The HRP-labeled anti-immunoglobulingoat antibody (1:5,000 dilution in blocking solution) was then allowed to react for 1 h at room temperature with gentle shaking. The membrane was again washed as after primary antibody reaction. Colour development reaction was carried out by immersing washed membrane in a 10 ml solution containing 10 mg DAB. 10 mg imidazole and 1-2 µl H2O2 The development reaction was stopped by washing the membrane with ample quantity of distilled water Analysis of oxidized samples Aliquots of oxidized samples were centriftiged at full speed (14.000 rpm) for 10 min at room temperature in a microcentrifuge, the supernatants were collected, mixed with 10 mM EDTA (final concentration) and analysed further. Protein estimation The protein retained in the supernatant (of 14,000 rpm for 10 min) after oxidation at different initial protein concentrations, at different GdCI concentrations and for different duration were estimated using Bradford's method (Bradford. M.M.. 1976.. Anal. Biochcm. 72: 248). Analytical high performance liquid chromatography (HPLC) The reverse phase HPLC (RP-HPLC) which distinguishes between the reduced and unreduced bGH as described earlier (Langley. K.E.. Lai, P.H., Wypych, J., Everett. R.R.. Berg. T.F., Krabill, L.F., Davis. J.M. and Souza, L.M., 1987., Eur. J. Biochem. 163: 323), was used here with modifications. The analysis were carried out using Applied Biosystems Model 172A HPLC system. A C-4 RP-HPLC column (Matrex silica, Amicon, USA; 4.6 mm x 100 mm column dimension; 6.5 µ particle size and 250A pore diameter) was used at a constant flow rate of 300 µl/min. With solvent A (0.1% TFA in HPLC grade water) and B (0.1% TFA in HPLC grade acetonitrile), a step gradient was applied over a total period of 50 min as follows: Step 1 : at 0% B hold for 1 min Step 2 : from 0% B to 10% B over a period of 4 min Step 3 : from 10% B to 55% B over a period of 15 min Step 4 : from 55% B to 70% B over a period of 25 min Step 5 : from 75% B to 100% B over a period of 5 min The protein samples were injected in a total volume of 80µ1 containing 2% TFA (final concentration). 25 µg of either unreduced (unreduced std. pit. bGH or air-oxidized rGH in 10 mM EDTA) or reduced (with DTT, of 20 mM final concentration at room temperature for 30 min. just before HPLC analysis). The HPLC elution profile of unreduced and reduced pituitary bGH served as the standard for monomenc oxidized and reduced form or GH respectively. The data acquisition and analysis was done by ABI 600 Data Analysis System. The area under oxidized monomer, reduced and wrongly oxidized (see section 3.3.4.2 in the Discussion) peaks were expressed as per cent of the total area. Non-reducing SDS-PAGE The oxidized samples (in 6 M GdCl) as well as oxidized (unreduced) pituitary bGH standard were precipitated by 10% TCA (to 800 µl of protein sample was added 200 µl of 50% TCA. mixed, kept on ice for 30 min and pelleted by centrifugation for 10 min at 14.000 rpm in a microcentrifuge at 4°C). The pellet was washed with 500µl of 80% acetone containing 10 mM EDTA. Air dried pellet was dissolved in IxMSB (without any reducing agent) containing 10 mM EDTA. Samples were resolved similarly on SDS-PAGE as described in appendix, except 10 mM EDTA was also incorporated in the gel as well as in the running buffer. Thiol analysis Analysis for free thiol (-SH groups) was done by the method of Ellman (Ellman, G.L., 1959.. Arch. Biochem. Biophys. 82: 701959) as described by Habeeb (Habeeb, A.F.S.A.. 1972, Methods Enzymol. 25: 4571972). Approximately 300 µg of protein (-0.01 µmol) was diluted to 1ml in which final concentrations of GdCl and EDTA, were 5 M and 0.5 mg/ml respectively. The addition of SDS was omitted to avoid precipitation and 50 mM Tris HC1, pH 8.5 was used instead of 0.1 M phosphate buffer. To protein solution DTNB (from a stock of 4 mg/ml) was added to a final concentration of 133.33 µg/ml and the reaction was allowed to proceed for 30 min at room temperature. Apparent absorbance was taken at 410 nm in a Shimadzu Spectrophotometer against a protein blank (without DTNB). The net absorbance was obtained by subtracting the absorbance due to reagent blank (without protein). For calculation of free sulfhydryl groups, the net absorbance was employed with a molar absorptivity value of 13.600 M-1 cm-1. Receptor binding assays Preparation ofmicrosomal membrane fron(goat liver The GH receptor was prepared from liver membrane of goat following the process described earlier (Haro, L.S., Collier, R.J. and Talamantes. F.J.. 1984.. Mol. Cellular Endocrinol 38: 109) with modifications. Goat liver was collected within 5 min of slaughter in an ice bucket and processed further within 15 min of collection. Approximately 20 g of liver tissue was chopped into fine pieces with a scissors and homogenized in 100 ml of Tris buffer (25 mM Tris HC1, 10 mM EDTA. 10 mM EGTA) containing 300 mM sucrose. ImM PMSF. 20 ug/ml Soyabean trypsin inhibitor. pH 9.0. In a glass homogenizer 20 strokes were given with a pinch of glass powder added to the sample. The homogenate was subsequently centrifuged at 10,000 x g for 15 min at 4°C. The supernatant was further centrifuged at 1.00,000 x g for 2 h at 4°C with a SW-28 rotor. The pellet of 1,00,000 x g was resuspended in 20 ml of Tris buffer containing ImM PMSF, pH 7.7 with 10 strokes in a glass homogenizer. This membrane suspension was stored at -70°C in aliquots of 1ml till further use. lodination of GH The pituitary bGH was iodinated by the 'lodo-gen™' method as described (Cadman, H.F. and Wallis, M., 1981, Biochem. J. 198.605). lodogen coated vial was prepared by drying 50 µl iodogen solution (of 0. Img/ml in chloroform) in a thin film with gentle jet of gaseous nitrogen in a small polypropylene tube. About 10 ug hormone (standard pituitary bGH, National Institute of Health. USA) in a total volume of 30 ul in phosphate buffer (0.5 M NaH2PO4, pH 7.4) was added to the iodogen coated vial and kept for 5 min with frequent gentle tapping at room temperature. To it 1 mCi 125I (in a total volume of 20 µ) was then added and the content was thoroughly mixed. The labeling reaction was allowed to proceed for 10 min at room temperature with intermittent tapping. The reaction was diluted with 200 µl of phosphate buffer, pH 7.4. The diluted labeling reaction was then carefully transferred onto an already equilibrated (with 5 bed volumes of buffer containing 25 mM Tris HC1. pH 7.5, 0.05% BSA and 0.05% Na-azide) Sephadex G-75 (regular grade) column (0.75 cm x 12 cm; with approximately 5 ml bed volume). Fractions of approximately 0.5 ml were collected and the counts (CPM) of 10µl aliquots of each fractions were measured in a Cobra'- (Packard) automated gamma counter. The TCA precipitable counts of peak fractions were also taken [10 µl of sample was precipitated with 10% (final) TCA in a total volume of 1 ml with added BSA (1 mg/ml) for 30 min on ice followed by centrifugation at 14,000 rpm in a microcentrifuge for 10 minutes]. Setting up of assay A competitive radio-receptor assay (RRA) was carried out using 25 ul of membrane preparation (150µ,g of protein), and 10.000 CPM of radio-labeled bovine pituitary GH (125I. bGH) in a final volume of 500µl in assay buffer (10 mM EDTA. 10 mM EGTA. 0.1% BSA. 0.05% Na-azide and 25 mM Tris HC1. pH 7.6). The 125I-bGH and different amounts (0, 0.1,4, 10. 100, 1000 and 10000 ng) of cold (unlabeled) hormone was simultaneously allowed to bind to the receptor for 4-5 h at room temperature. The binding reaction was stopped by adding 1ml of ice-cold assay buffer and the membrane (receptor-ligand) complex was settled by centrifugation at 5000 rpm for 20 min. All traces of supernatant was removed carefully. The membrane bound radioactivity was counted in a Cobra'- (Packard) automated gamma counter. The total binding (Bt) and non-specific binding (Bn) were obtained respectively from the reactions where there was no cold hormone added and very high amount (10.000 ng) of respective cold hormone was added. However, the specific binding (Bs) in each reaction was indirectly calculated by subtracting the non-specific counts from the respective pellet-associated counts. Sequences of various oligonucleotide primers I. RT-PCR primers a) 1st strand cDNA synthesis primers i) Oligo-dT primer [Supplied with Stratagene's first strand synthesis kit] b)PCR primers i) BGH1 [Kpnl primer] Kpnl Hybridizing area (Sequence Removed) ii) BGH2 [BamHI primer] BamHI Hybridizing area > (Sequence Removed) 2. Sequencing primers a) Forward [Ml3 universal sequencing primer of USB/Amersham] (Sequence Removed) b) Reverse sequencing primer (Sequence Removed) c) UM1 [Gro\vth hormone sequence specific primer. This primer binds on GH sequence 3'- end of which is about 100 base away from 'A' of start codon ATG.] (Sequence Removed) 4. Oligos used to constnict s>nthetic first cistron of a two-cistronic expression system a) BGHB1 (Sequence Removed) b) BGHB2 (Sequence Removed) Bacterial strains All the bacterial strains used in this study are listed in the table A.I (below). The E. coli strains were maintained on LB plates at 4oC for short term storage. For long term storage, cultures were frozen in 25% glycerol at -70 °C. Table 1 Standard E. coli strains used. (Table Removed) EXAMPLES Example 1: The isolation and cloning of genetic sequence encoding GH (GH-cDNA) polypeptide/s from Indian species of cattle (Bos indicus). buffalo (Bnbains bitbalis) and goat (Capra hircus) was carried out using rDNA techniques as follows. The cDNA spercies specifying the growth hormone polypeptide/s corresponding to the different animals were isolated by the reverse transcriptase polymerase chain reaction (RT-PCR). This technique (Veres. G.. Gibbs. R.A.. Scherer. S.E. and Caskey. C.T.. 1987.. Science 237: 415) directly converts a specific mRNA into double stranded cDNA followed by its amplification by PCR. Thus, highly specific enriched molecules generated by this technique make the overall procedure almost akin to a subcloning, virtually eliminating the cost and cumbersomeness of screening a library of clones. The entire process of isolation of the requisite genetic determinant/s and boarding them onto an self-replicating vector DNA molecule (replicon) for their independent propagation was carried out by the following discrete steps. The total RNA was isolated by single-step guanidinium-thiocyanate-phenol-chloroform method (ChomczynskL P. and Sacchi. N.. 1987.. Anal. Biochem. 162: 156) using the pituitary-tissue of freshly slaughtered animals. There are several alternative methods for isolating RNA from animal tissues as described (AusubeL F. M.. Brent. R.. Kingston. R. E.. Moore. D. D., Seidman, J. G.. Smith. J. A. and Struhl. K... 1987. 'Current Protocols in Molecular Biology' Green publishing associates and Wiley-Interscience: Sambrook, J., Fritsch. E.F. and Maniatis, T.. 1989. "Molecular cloning a laboratory manual". Cold Spring Harbor Laboratory Press). Isolation of RNA was carried out using Stratagene's RNA isolation kit™. The pre-weighed frozen tissues was pulverized in a pestle and mortar and finally homogenized in an Omni mixer. With smaller quantities of tissues, as with goat pituitary, the frozen tissues were directly pulverized into fine powder in a pestle and mortar in presence of liquid nitrogen. Isolated RNA was resuspended in DEPC treated water. First strand cDNA was synthesized upon total RNA using random primers. Moloney Murine Leukemia Virus (MMLV) reverse-transcriptase (RT-ase) was used to catalyse the reverse transcription reaction. PCR amplification of first strand reaction product (Figure 1) was carried out by Perkin Elmer Cetus's Gene Amp™ DNA amplification reagent kit and its DNA Thermal Cycler. PCR parameters were: Initial denaturation at 94°C for 5 mm followed by 25 cycles of amplification with denaturation temperature of 92°C. annealing at 55°C and extension at 72°C, 1 min each, followed by a 5 min final extension step. A pair of GH specific primers viz., BGH1 and BGH2 (see "General Method' section for sequence) were used for the PCR. These primers were designed based on the published GH mRNA sequence of taurine cattle (Miller. W.L., Martial. J.A and Baxter. J.D., 1980. J. Biol. Chem., 255: 7521). Pfu UNA polymerase was used to catalyst the PCR reaction. The first strand reaction product was subsequently amplified using Pfu DNA polymerases. For all the species studied the RT-PCR using a pair of bGH sequence specific primers resulted in a single species of amplified DNA molecule co-migrating (as expected) with the 564 bp band of Hindlll digested λ-DNA marker (see Figure 1 and Figure 2A and B). The PCR product was cloned using the previously described method (Jung, V., Pestka. S.B. and Pestka. S.. 1990.. Nucl. Acids Res. 18: 6156). The PCR product was purified using QIAGEN spin-20™ (Qiagen Inc.) to remove excess primers, unincorporated nucleotides. enzyme and mineral oil. The above reaction mixture was directly used for kinasing without further purification by T4 PNK. Initially an insert-insert intermolecular ligation reaction was carried by T4 DNA ligase using approximately 1µg of kinascd PCR product in 10 µl volume. After heat inactivation of ligase the above reaction was directly used for restriction digestion by Kpnl and BamHl under optimal conditions of buffer, enzyme and temperature. Approximately 5µg of purified plasmid pBluescript II KS" DNA was sequentially digested by Kpnl and BamHl under optimal conditions of buffer, enzyme and temperature. The digested insert and vector DMAs were purified from 1.2% and 0.8% agarose gel respectively using Biorad's Prcp-A-Gene™ kit. Finally, vector-insert ligation and subsequent transformation into competent E. coli XL 1 Blue cells were carried out as described (Sambrook. J.. Fritsch. E.F. and Maniatis. T.. 1989.. "Molecular cloning, a laboratory manual". Cold Spring Harbor Laboratory Press. New York ). This method yielded 4.3x105 white clones per microgram of vector DNA used, under conditions where supercoiled vector DNA (uncut control plasmid) gave an efficiency of about 108 transformants per microgram. Clones were cofirmed by restriction endonuclease digestion and selected clones were found to carry inserts of expected size. Example 2: Determination of nucleotide sequence of the cloned genetic material (GH cDNAs) specifying GH-pol\peptide/s was carried out by Sanger's dideoxy chain termination method (Sanger, F.. Niklenr S. and Coulson, A.R.T Proc. Natl. Acad. ScL USA. 74: 5463). The sequencing reactions were c.tHed out (using double stranded plasmid DNA as template) by USB's Sequenase'8' version 2.0 DNA sequencing kit. Two clones of GH cDNA of each species were subjected to complete nucleotide sequencing. Either cesium chloride - ethidium bromide density gradient purified or miniprep plasmid DNA was used for sequencing. Mimprep DNA was further purified using PrepA gene™ kit (BioRad Inc.. USA). Approximately 0.5 pmol of M13 universal forward and reverse primers (USB/Amersham) were used. All the sequencing reactions were resolved either on 6% or 8% urea (7 M)-acrylamidc gel using LKB2010 T\f Macrophor electrophoresis unit (LKB). A total of 573 bases sequence, encoding 191 amino acids, of all the three species were unambiguously determined. The GH cDNA sequence between clones (descendant of independent PCR reactions) in each species were found identical. The compiled sequences revealed several nucleotide differences among the GH-cDNA of these species and also as compared to some of the know:n closely related ruminant animal GH sequences (Figure 3 A & B). Example 3: Hyper-expression of GH-cDNAs in E. coli \vas achieved through a two-cistronic strategy (Schoner. B.E.. Belagaje. R.M. and Schoner. R.G.. 1986.. Proc. Natl. Acad. ScL USA. 83: 8506). For this purpose two synthetic oligos BGHB1 (55-mer) and BGHB2 (51-mer) were designed so that upon annealing they would form a short synthetic cistron with a 8-amino acid coding sequence and ending with a stop codon. This synthetic cistron also contained a RBS located at the 3'-end of the cistron . This downstream RBS is designed to be used by the second cistron (GH gene). The GH cDNA was spliced downstream to the synthetic cistron and the entire two-cistronic cassette was subcloned under the trc promoter in the vector pTrc99A (Figure 15). The nucleotide sequencing of the N-terminal region of GH gene, as well as entire synthetic first cistron of of two-cistronic construct was carried out using GH sequence specific primers prior to expression studies (Figure 4). The nucleotide sequence, of the N-terminal portion of the GH gene and the entire synthetic cistron revealed incorporation of the desired modifications with complete fidelity. The tentative level of expression was estimated to be in the range of 25-30%. when the post-induced culture was analysed on SDS-PAGE. To confirm the expression of GH. and also to determine the tentative level of expression, a quantitative Western blot was also carried out. where different amounts of standard bGH and post-induced lysate of pUG99II/B were ran in parallel. Quantitative analysis of such Western blots showed the expression level to be in the range of 50-100 mg GH/L of shake flask culture (of approximately 1 OD600 unit). Example 4: Expression of GH in different E. coli host strains of the two-cistronic construct pUG99II/B which was found to hyper-express GH in E. coli XL 1-Blue strain, was then carried out as follows. The plasmid was transformed through electroporation into four other commercially available E. coli host strains, namely BL-21. Toppl. Topp2 and Topp3 (see Table 1 for genetic attributes of these strains). The presence of the right construct among transformants was confirmed through restriction enzyme digestions with appropriate 'diagnostic' enzymes. It was observed that Topp2 strain attained the highest OD600 of 3.89 within 8 h of induction under shake flask conditions. The highest accumulation of rGH per unit cell mass (equivalent to 20µ1 of post-induced culture of 1 OD600 unit) for XL 1-Blue (2.4µg). BL-21 (2.5 µg). Toppl (2.5 µg). Topp2 (2.6 µg) and Topp3 (1.85µg) were observed at 16, 8, 8, 16 and 4 h after induction (Figure 5), respectively. However, an estimate (by densitometry) of rGH produced per milliliter of culture revealed that the Topp2 strain of E. coli is superior to all other strains tested in terms of total production of GH within 4 to 8 h of induction. Example 5: For the harvesting of rGHs from genetically modified E.coli strain, the inoculum was raised by inoculating 10 ml of LB-Ampicillin (100 µg/ml) media with freshly grown single, well-isolated colony (~2 mm diameter) from agar plate and grown overnight (12-15 h) at 37°C with shaking (200 rpm). Next day 1 L of LB (with 50 ug/ml Ampicillin) in 5 L Erlenmeyer flask was seeded with overnight grown culture at 0.5% level and grown upto an OD600 of about 0.5. At this point the culture was induced with ImM (final concentration) IPTG and grown further for another 6-8 h. with vigorous shaking (200 rpm) at 37°C. Cells were chilled on an ice-water bath and were subsequently harvested by centrifugation at 5000 x g for 10-15 min in GS-3 rotor (Sorvall) at 4°C. The cell pellet was either processed immediately or frozen at -70°C. The 1L culture of 8 h growth (post-induction) reached an OD600 of nearly 3.0, yielding 3.5-4.0 g wet weight of cells (Topp2 strain of E. coli containing construct pUG99II/B). Cell pellet obtained from upto 100 ml culture was lysed by the physical method as previously described (Schoner, R.G., Ellis. L.F. and Schoner, B.E., 1985., Bio/Technology, 3: 151), alternatively, the higher quantities of cells (from 1L cultures) was lysed chemically (Harris, T.J.R., Patel, T., Marston, F.A.O., Little, S., Emtage, J.S., Opdenakker, G., Volckaert, G.. Rambant, W.. Billiau. A. and De Somer, P., 1986., Mol. Biol. Med. 3: 279) as follows: i) For each 1 g wet weight of cells 9 ml of lysis buffer (as above) was added and cells were resuspended by vigorous vortexing. ii) To the cell suspension was added 10 µ1 of 50 mM PMSF (freshly prepared in 100% methanol), followed by 250µl of lysozyme (10 mg/ml. freshly prepared in lysis buffer). iii) This suspension was incubated on the ice for 30 min with occasional mixing, iv) Then. 10 mg DOC was added with stirring. v) When the suspension became viscous. 100µl DNAase (1 mg/ml. freshly prepared in lysis buffer) was added with stirring. vi) Then the suspension was incubated at room temperature until it was no longer viscous (about 1 h). The efficiency of lysis was checked under the light microscope for the presence of intact cells before and after lysis. The lysis of cells was found optimal only when the cell pellet was dissolved at a ratio of 10:1 or more (milliliter of lysis buffer per gram wet weight of cells) both in case of mechanical (Schoner, R.G.. Ellis. L.F. and Schoner. B.E.. 1985. Bio/Technology. 3:151) and chemical lysis (Hams, T.J.R., Patel. T., Marston, F.A.O.. Little. S., Emtage, J.S.. Opdenakker, G., Volckaert. G., Rambant. W., Billiau. A. and De Somer. P.. 1986. Mol. Biol. Med., 3: 279). Overall, sonication was found superior to the chemical lysis method, particularly in case of smaller volume of cells. It was found that the increased viscosity of the lysate resulting out of chemical lysis of cells was difficult to reduce using even high concentrations of DNAase (100 µg/g cells) and longer incubation time (2 h) than recommended (20 µg DNAase/g cells and 30 min respectively). To overcome this problem samples were either sonicated briefly or 10 mM magnesium chloride was added during DNAase treatment. GH-IBs were differentially sedimented from complex mixture of E. coll proteins, i.e.. caide cell lysate. Initially smaller aliquots of crude lysate (100µ1) were spun under different r.c.f (relative centrifugal force) in a Haerius microcentrifuge at 4°C for 15 mm to study the sedimentation behaviour of the IBs. The various r.c.f values tried were. 50. 100. 200. 300, 500. 1000. 2500. 5000 and 10.000 x gs. The supernatant was carefully collected and mixed with 100 µl of 2xMSB, whereas the pellet was first suspended in 100 µl water before adding equal volume of 2xMSB. Both the pellet and the supernatant were subsequently processed and analysed on SDS-PAGE. It was found that the GH-IBs sedimented at as low as 200 x g (Figure 6 A). Most of the IBs sedimented at 1000 x g and complete sedimentation occurred at 5000 x g within 10 min. The crude IB-preparation was first washed with equal volume of water by resuspending pellet (from 100µl crude lysate) in 100µ1 water, followed by centrifugation at 10,000 x g for 15 min at 4°C. The water washed pellet was further washed with the following agents of different concentration, individually or in combination: i) Urea washing with 1T 2. 3. 4. 5. 6 and 7 M urea ii) 0.1M salt (NaCl) in 0.1M Tris HC1, pH 8.5 iii) lOmMEDTA iv) 0.5% Triton X-100 v) Salt (0.1M NaCl in 0.1M Tris. pH 8.5) in combination with 4. 5 and 6 M urea. vi) Triton X-100 (0.5%). EDTA (10 mM) in combination with 2. 3. 4 and 5 M urea. For all the above washing treatments. 100 µl of water washed pellet was resuspended in 100µl of respective reagents, incubated at room temperature for 15 min with gentle shaking followed by centrifugation at 10.000 x g for 15 min at 4°C. The resulting supernatant was carefully collected and was directly mixed with 2xMSB (100µl) whereas pellet was resuspended in 100 µ1 water before mixing with 2xMSB (100µ1). These samples were further processed for analysis on SDS-PAGE. The water-washed pellet was found to contain nearly 75-80% GH. with the rest (20-25%) being composed of contaminating E. coli proteins. The 5 M urea washing removed another 20-30% of the contaminating proteins. No dissolution of GH-IBs was observed upto 5 M urea washing, whereas GH started leaching out in the supernatant at 6 M urea washing (Figure 6B). Washing with various other agents like EDTA. deoxycholate and salt (different molar NaCl) were not found to be effective in removing contaminating proteins, whereas 0.5% Triton X-100 was found to be the most effective cleaning agent (even better than 5 M urea). Therefore, a combination of different molar urea with 0.5% Triton X-100 was used to further enhance the purity of GH in IB-preparation. It was found that GH started appearing in the supernatant at 4 M urea -Triton X-100 (0.5%) washing (Figure 6 C). The 3 M urea-Triton X-100 (0.5%) combination, was found to be the best washing condition completely eliminating the major contaminating proteins with a negligible loss of GH (Figure 6 C. lane 1 and 2). For preparative purposes water washed IBs were further cleansed with a solution of 3 M urea. 10 mM EDTA and 0.5% Triton X-100. in 0.l M Tris HC1, pH 8.5. Example 6: In order to earn out the dissolution of inclusion bodies, the pellets of purified IBs were dissolved at different temperatures (either 55°C. 25°C or 4°C) by resuspending the pellets at 2.5 mg/ml (final protein concentration) in different concentrations of urea (6. 7 and 8 M urea in 0.1M Tris HCI. pH S.5). The suspension \vas then incubated ut room temperature for 30 min with gentle shaking (100 rpm). The various final protein concentrations (1. 2.5. 5 and 7.5 mg/ml) were also studied similarly using 8 M urea (in 0.1M Tris HCI. pH 8.5) at room temperature. IBs were also dissolved using 6 M and 8 M GdCl (in 50 mM Tris HCI. pH 8.5) at the protein concentrations of 5. 10 and 15 mg/ml at room temperature for 15 minutes. The 7 M urea was able to dissolve only upto 1 mg/ml IBs. whereas 8 M urea dissolved upto 2.5 mg/ml IBs across all the temperatures (4, 25 and 55°C) studied. We found GdCl to be a much more potent dissolving agent than urea, as 6 M and 8 M GdCl was found to completely dissolve 10 and 15 mg/ml IBs. respectively, at room temperature within 5 minutes. For subsequent oxidation studies. IBs were routinely dissolved using 8 M GdCl in 50 mM Tris HCI. pH 8.5. at room temperature for 5-10 min upto a final protein concentration of 10 mg/ml. Example 7: GdC!-sclubilised GH was air-oxidized using simple protocol in an open vessel at room temperature with gentle stirring in absence of any reducing agent and without the use of any oxidation facilitating agents. Oxidation kinetics of goat GH was studied under the following conditions: i) A protein concentration of 1 mg/ml was oxidized at various (final) GdCl concentrations (2. 3. 4. 5 and 6 M) for 72 hours. ii) Samples with various starting protein concentrations (0.25. 0.5. 0.75. 1.0. 1.25. 1.5. 2.0 and 2.5 mg/ml) were oxidized at a GdCl concentration of 6 M for 72 hours. iii) Oxidation was also carried out for different time periods (0. 1. 3. 5, 7. 9. 11. 13, 24. 36. 48 and 72 h) with 1.5 mg/ml starting protein concentration and at 6 M GdCl in 50 mM Tris HCI. pH 8.5. For analysis, the oxidation was stopped by adding ETDA (to a final concentration of 10 mM) to aliquots of oxidation reaction mixtures and stored at -70°C until further analysis. The air-oxidation of thiol groups of protein is catalysed by metal-ions. However, no metal-ion was externally added as the trace amount of metal-ions required for oxidation was thought to be available in the low-grade GdCl used in the reaction. The oxidation was carried out under different conditions and for different durations to identify the most optimal conditions to maximise the final yield of correctly folded, bioactive rGH. To distinguish between oxidised and reduced form of GH we have used a combination of RP-HPLC and SDS-PAGE (non-reducing) techniques as has previously been shown (Langley. K.E.. Lai. P.H., Wypych. J., Everett, R.R.. Berg, T.F., Krabill, L.F.. Davis. J.M. and Souza, L.M., 1987. Eur. J. Biochem, 163: 323; Pint N.K., 1991., FEBS Lett. 292: 187). We have also found that standard oxidised (i.e. unreduced) and reduced (see: General Methods used in Examples) pituitary bGH to" have difference in retention time (Rt) on RP-HPLC and mobility on non-reducing SDS-PAGE (Figure 7). Oxidative-refolding of solubilised rGH obtained from IBs was carried out at different protein concentrations varying between 0.25 mg/ml to 2.5 mg/ml in 6 M GdCl at room temperature for 72 h (see: General Methods used in Examples). At the end of the raection. samples were subjected to high-speed (10.000 x g) centrifugation for 10 min to eliminate any insoluble aggregates formed during the course of oxidation. The resulting supernatant was analysed for protein content to assess the loss of protein due to aggregation during oxidation. The soluble protein was then analysed by RP-HPLC and SDS-PAGE (non-reducing) to measure the abundance of the desired form of the polypeptidc (correctly oxidised and refolded monomer). The estimation of protein retained in the supernatant revealed that about 20-25% of the initially soluble protein disappeared during oxidation in case of the lowest starting protein concentration tested (0.25 mg/ml). The loss was found to decrease progressively with increase in starting protein concentration in the oxidation reaction, with virtually no loss being observed at or above the protein concentrations of 1.5 mg/ml. The RP-HPLC analysis of equal amount of soluble protein (i.e. soluble in 6 M GdCl) from all the oxidised samples showed that the relative area under the peak corresponding to that of oxidised monomer in case where oxidation was carried out at 2.5 mg/ml concentration was approximately 80% of that obtained when oxidation was carried out at a protein concentration of 0.25 mg/ml (taken to be composed of 100% oxidised monomeric form of GH as it was found to give qualitatively the 'cleanest' oxidation product with no other form being observed) [see Figure 7 and 8]. The GdCl which was found to be an efficient dissolving agent of rGH-IBs was also used as medium for carrying out subsequent oxidation of rGH molecules, most of which were found to be in the reduced form. However, a very high concentration of the chaotropic agent could be too denaturing to allow the protein folding and disulphide bond formation. On the other hand, a low concentration, although it may permit the protein to fold back to its native conformation, but may not prevent intermolecular aggregation specially under circumstances where the oxidation is carried out at higher protein concentration and the given protein has high innate propensity for intermolecular aggregation even under moderate protein concentration. Therefore, both too high or too low concentration of the GdCl could be detrimental to the overall yield of the correctly folded/oxidised monomeric form of the target polypeptide. Accordingly, air-oxidation of rGH at different molar GdCl (2, 3. 4, 5 and 6 M) with a fixed (1 mg/ml) starting protein concentration (at room temperature for 72 h) was carried out to find out the most optimal GdCl concentration which would allow maximum final yield of correctly folded/oxidised monomeric form of the soluble protein. It was found that loss of protein during oxidation, possibly due to the formation of insoluble aggregate, was very high at GdCl concentration of 2 M. However, virtually no loss of protein due to aggregation was observed at GdCl concentration of 6 M. Equal amounts (equivalent Bradford color value) of soluble protein fraction (i.e. protein retained in the supernatant after 72 h of oxidation reaction) were then analysed by RP-HPLC and non-reducing SDS-PAGE for the qualitative and quantitative assessment of the oxidation end-products. It was found by RP-HPLC analysis that the relative area (per cent of total) under oxidised peak was the highest in case of 2 M GdCl concentration with virtually all the protein of the soluble fraction eluting under a single peak and no other peak being observed. The reduced form was found to disappear across all the GdCl concentrations tested after the oxidation reaction; either no peak (in case of 2, 3. and 4 M GdCl) or a very small peak (contributing only about 0.5 and 1.5% of the total area in case of 5 and 6 M GdCl respectively) at the region of reduced form was observed. However at an intermediate GdCl concentration of around 4 M (RP-HPLC profile not shown) an additional prominant peak (with Rt of about 33 min) eluted after the oxidised (Rt=27 min) and reduced (Rt-30 min) forms (all the three forms/peaks can be see in panel E of Figure 7). This peak was presumed to be formed due to wrongly oxidised form of GH (scrambled GH) and constituted about one fourth of the total oxidation product at this concentration of GdCl. The overall final yield of oxidised monomeric form of rGH at the end of oxidation reaction was found to be the highest (about 90%) at GdCl concentration of 6 M. The oxidation kinetics of rGH was studied by analysing samples removed at different time points (0. 1. 3. 5. 7, 9. 11. 13. 24. 36. 48 and 72 h) by RP-HPLC as well as non-reducing SDS-PAGE. It was found that the starting material (0 h sample) had about 80% of GH in the reduced form (Figure 7 C and Figure 11 A, lane-2). As the oxidation proceeded, the relative area ( per cent of total area) under reduced peak progressively decreased and became negligible after 36 h of oxidation (Figure 7 E and Figure 11 A, lane-4). In parallel, the samples were also analysed by non-reducing SDS-PAGE for cross-validation of the relative abundance of oxidised and reduced forms of rGH. In the Figure 11 A, the oxidized samples of 0, 3 and 36 h (which respectively had about 20, 50 and 100% oxidized form as per HPLC analysis) are shown. The samples obtained at different durations during oxidation were also subjected to Ellman's reaction for the estimation of free thiol groups. As can be seen (Figure 8) there was a sharp reduction of'SH-groups' within 10-12 h of simple air-oxidation and no free thiol could be observed in samples oxidised after the initial 36 hours. The data presented shows a clear concordance between the values of the relative abundance of reduced and oxidised forms of rGH during various stages of oxidation when measured by different biophysical and biochemical methods. Example 8: Chromatographic purification of the various rGH proteins to homogeneity was carried out to obtain pure hormones. Binding characteristics of GH was initially checked with Phenyl-Sepharose (Pharmacia Ltd.), Hexyl-agarose and Decyl-agarose (Affinity Chromatography Ltd., U.K.) using either different molar NaCl (0.25, 0.5. 1.0 and 1.5 M) or ammonium sulphate (0.5, 1.0, 1.5 and 2.0 M) as the binding buffer. Mini-scale binding studies were first carried out using 100 µl equilibrated beads ( with 50 mM Tris HC1 pH 7.5 containing respective molarity of salt) and 100-200µg of protein (oxidized sample) in a final volume of 500 µI at room temperature for 30 minutes. Binding of GH onto Decyl-agarose was also studied either in presence of different amount of GdCl (6, 5. 4. 3. 2.5 and 2 M) at pH 7.5 or at different final pH (9.5. 9.0. 8.5. 8.0. 7.5 and 7.0) in presence of 2 M GdCl. For preparative purpose 10 mg of oxidized sample was bound to 10 ml (volume of swollen beads) of Decyl-agarose as follows: i) In a 250 ml beaker 10 ml of equilibrated (with 2 M GdCl. 50 mM Tris. pH 7.5) resin (Decyl-agarose) was taken and placed on a magnetic stirrer at room temperature. ii) Approximately 7 ml of oxidized protein sample (1.5 mg/ml protein in 6 M GdCl, 50 mM Tris. pH 8.0) was added on the resin while stirring was still going on. iii) Simultaneously and rapidly 14 ml of 50 mM Tris HC1. pH 8.0 was also added (to bring down the final GdCl and protein concentration to 2 M and about 0.5 mg/ml respectively) and the stirring was further continued (at a sufficient speed to keep beads in suspension) for 5 minutes. iv) A 21 ml of a diluent (containing 1M ammonium sulphate. 50 mM Tris HCI pH 7.5) was then added rapidly. This brought down final GdCl and protein concentrations below 1M and 0.25 mg/ml respectively. However the stirring was continued for another 30 minutes. v) The protein content of the supernatant was determined and the resin was loaded onto a 8.5 x 1.5 cm column, under gravity. vi) The column was then washed with 3 bed volume of 0.5 M ammonium sulphate in 50 mM Tris HCl. pH 7.5 to remove the unbound proteins. vii) The column was further washed with 5 bed volumes each of 50 mM Tris HCI (pH 7.5). distilled water and 8M urea (in water) at a flow rate of 30-40 ml/h. viii) Fractions of approximately 5 ml volume were collected and the protein content was analysed by Bradford's method (Bradford. M.M..1976. Anal. Biochem.. 72: 248). Approximately 11 mg oxidised protein was allowed to bind to Decyl-agarose as described above. About 80% (8.75 mg) of the loaded protein was found to be captured by the resin as estimated by measuring the protein content of the supernatant. A total of 7.95 mg (71% of bound protein) was recovered in which 8 M urea represented about 6 mg protein (Figure 9) with nearly 90% purity (see Figure 12? lane 4-7). The other peak which had eluted in water was found to contain contaminating E. coli proteins with no visible band parallel to GH being observed. The overall yield of rGH at the end of the HIC purification was 60% of the initial amount. The HIC-purified material was further purified by ion-exchange chromatography onto DEAE-Sepharose (Fast-flow). A 10 ml (volume of swollen beads) column was packed, regenerated with three bed volumes of 1M NH4HC03. pH 9.5. followed by equilibration with five bed volumes of 5 mM NH4HC03, pH 9.5 (pH adjusted with liquid ammonia). The materials of two peaks (8 M urea peak) of the HIC (above) were pooled and approximately 10 mg of protein sample was adjusted to 5 mM NH4HC03. This sample was passed through the column at a flow rate of 20-25 ml/h. the column was washed with three bed volume of 5 mM NH4HC03 (pH 9.5) to wash off the unbound proteins. The bound proteins were eluted with a linear salt gradient (5 mM-lM) of NH4HC03 (pH 9.5, adjusted with liquid ammonia) in a total volume of 100 ml. The fractions of approximately 4 ml volume were collected and analyzed for protein content. Approximately 90% (9.2 mg) of the loaded protein was found to be bound to the column as estimated indirectly by measuring the protein content of the flowthrough. The total recovery from the IEC column after elution with a salt gradient was over 88% (8 mg) of the bound protein with about 65% (5.8 mg) of the total rGH loaded being recovered under a major peak (Figure 10). The GH peak eluted at around 400 mM NH4HC03 concentration. There was no other major peak observed other than GH. The overall yield of rGH at the end of IEC was 38% of the starting material (see Table 2 above), showing >98% purity (Figure 12, lane: 8-11) by SDS-PAGE (the values indicating approximate homogeneity of GH after each purification steps are based on densitometric scanning of samples resolved on SDS-polyacrylamide gel). The HPLC and non-reducing SDS-PAGE (Figure 11 A and B) analyses of the DEAE-purified material revealed the purification of highly enriched form of monomeric rGH. Example 9: Authentication of purified recombinant polypeptides by N-terminal sequence analysis was carried out. Approximately 20 µg of purified protein was resolved by SDS-PAGE and blotted onto PVDF membrane. This was then scqucnccd by gas phase Edman's sequential degradation (Edman, P. and Begg, A., 1967., Eur. J. Biochem. 1: 80) reaction in ABI's (Applied Biosystems Inc.. CA, USA) model 476A Protein Sequencer System. Reaction was run for 10 cycles. The HPLC profiles of phenylthiohydanation-amino acids (PTH-AA) released in each cycle were compared with standard PTH-AA profile. The data were analysed with ABI's 610A Data Analysis System. The N-terminal sequence of the first 10 residues of as obtained in an automated sequencer for GH of different species are presented in the form of a table. The HPLC profiles of first ten cycles (along with standard PTH-AAs profiles) of buffalo and goat rGH sequencing reactions are presented in Figure 13 A and B. The data also show that the recombinantly produced GH polypeptides were correctly translated with the N-terminal methionine (initiator methionine) being completely processed by aminopeptidases in expression host. Like other polypeptide hormones GH binds to specific cell-surface receptor to carry out its diverse biological functions and this forms the basis of its biological assay. The crude membrane preparation of various tissues from different species have been shown to bind 125l-labeled hGH (Posner N.. 1974.. Endocrinology 95: 521), however liver cell membrane has been shown to be the richest source of GH-receptor (Tushima. T. and Friesen. H.G.. 1973., J. Clin. Endocrinol. Metab. 37: 334). In this assay the bioactivity of the hormone molecule is tested by its ability to either inhibit the binding or to displace the bound tracer (which is usually a radio-labeled bioactive analogue of the hormone molecule). The radio-receptor assay (RRA) has been found to be an quicker and sensitive (more than bioassay) alternative to the bioassy (i.e., animal assay/growth promotion assay). The peak fractions (fraction no. 8. 9 and 10 with respective counts of 10 \i\ aliquot being 1238556, 1930092 and 1312576 CPM) of Sephadex G-75 chromatography of 125I labeled pituitary bGH were found to have high level of incorporated radioactivity (with TCA-precipitable count being about 85, 78 and 65% respectively. The fraction with least free label content was used subsequently for conducting assay. High non-specific binding (around 5000 CPM, where total binding was around 6000 CPM) were observed in all the cases. The results of competitive receptor binding assay is shown in the Figure 14. The rGH of buffalo and goat were found equally efficient as compared to pituitary bGH in inhibiting the binding of labeled pituitary bGH. The overall authenticity and integrity of the rGHs were validated based on the following biochemical, biophysical and biological criteria: i) The nucleotide sequence of the expression vector used for the production of rGH was found to harbour correct gene in right orientation and frame with required genetic elements for the expression of desired polypeptide. ii) The mobility of the rGH was found to be identical to that of standard pituitary bGH on reducing as well as non-reducing SDS-PAGE. iii) The rGH was also recognised by antisera raised against pituitary- bGH in Western blot analysis. iv) A sensitive analytical RP-HPLC technique standardized to distinguish between reduced and correctly oxidised monomeric GH molecules showed the finally purified material to have closely similar elution profile (retention time) to that of oxidised pituitary bGH standard (obtained from NIH. USA). v) Partial N-terminal amino acid sequencing data revealed the presence of correct amino acid sequence starting with 'alanine' with no N-terminal extension. No initiator methionine was detected in the HPLC-profile during amino acid sequencing indicating efficient processing of the formyl-methionine by the E. coli methionine- amino peptidases. Finally, in a competitive receptor binding assay using goat-liver receptor preparation, the rGHs were found to bind membrane-bound receptor with similar efficiency as chat of the pituitary bGH standard. We Claim: 1. A process for the preparation of recombinant growth hormone of important animals species with dairy and veterinary importance, useful for the enhancement of metabolic processes such as , enhanced lactation; said process characterized in that recombinant growth hormone being obtained by downstream splicing of growth hormone cDNA into a synthetic hyper expressive cistron synthesized from a two-cistronic expression systems no. BGHB1 and BGHB2 as described herein, and incorporating the entire two-cistronic cassette into a vehicle as describe herein to get hyper expressive growth hormone , the said process comprises: i) synthesis of the complementary DNA (cDNA) encoding for the growth hormone proteins of various animal species of veterinary importance, selected from the Indian water buffalo, Indian species of cattle (cow), goat, camel, donkey , yak either by chemical synthesis such as herein described , or by biochemical means using appropriate nucleic acid synthesizing enzymes as described herein , or a combination of these two methods, ii) recombining the growth hormone-encoding cDNA so obtained with a vehicle by downstream splicing of GH cDNA to a synthetic cistron as described above and cloning the vehicle in a heterologous host cell selected from a virus, bacterium, yeast, animal or plant cell to get recombinant vehicle carrying hyper expressive GH cDNA, iii) introducing the recombined vehicle so obtained into an appropriate heterologous host as defined above by conventional methods as described herein to get transformed cells, iv) isolating transformed cells carrying the hyper expressive growth hormone cDNA by conventional manner, v) expressing the growth hormones in the transformed host cells of step (iv) in an appropriate medium as described herein using conventional methods as described herein , vi) isolating the polypeptides of the growth hormones from the culture of the host cells and subjecting them to various fractionation and chromatographic methods as described herein to obtain highly pure desired growth hormones. 2. A process as claimed in claims 1 wherein the enzyme used for synthesizing the cDNA encoding for growth hormone proteins is DMA dependent RNA polymerase. 3. A process as claimed in claims 1-2 wherein the vehicle used for recombining the GH encoding cDNA is plasmid vehicle capable of either autonomous replication or after integration into the chromosomal DNA in a suitable host cell. 4. A process as claimed in claims 1 - 3 wherein the heterologous host cell used to express the growth hormone polypeptide is a virus, bacterium, yeast, animal or plant cell. 5. A process as claimed in claim no. 1 - 4 wherein conventional methods used to get the highly pure recombinant growth hormones is dissolution of polypeptides and then refolding by contacting and binding with a chromatrographic matrix containing hydrophobic groups such as methyl-, butyl-, hexyl-, phenyl- and decyl. 6. A process for the preparation of recombinant growth hormones from different animal species with dairy and veterinary importance substantially as described herein with reference to the examples and drawings accompanying this specification. |
|---|
1111-del-2001-correspondence-others.pdf
1111-del-2001-correspondence-po.pdf
1111-del-2001-description (complete).pdf
| Patent Number | 242286 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Indian Patent Application Number | 1111/DEL/2001 | |||||||||
| PG Journal Number | 35/2010 | |||||||||
| Publication Date | 27-Aug-2010 | |||||||||
| Grant Date | 20-Aug-2010 | |||||||||
| Date of Filing | 31-Oct-2001 | |||||||||
| Name of Patentee | COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH | |||||||||
| Applicant Address | RAFI MARG,NEW DELHI-110 001,INDIA | |||||||||
Inventors:
|
||||||||||
| PCT International Classification Number | A61D38/27 | |||||||||
| PCT International Application Number | N/A | |||||||||
| PCT International Filing date | ||||||||||
PCT Conventions:
|
||||||||||