|Title of Invention||
A CHIMERIC PROTEIN OF HUMAN GRANULOCYTE COLONY STIMULATING FACTOR
|Abstract||The present invention relates to a chimeric protein of recombinant human granulocyte colony stimulating factor comprising of hG-CSF fused to an affinity fusion tag at its amino terminus.|
THE PATENTS ACT, 1970 (39 of 1970)
(See Section 10; rule 13)
" A Chimeric Protein Of Human Granulocyte Colony Stimulating Factor "
RELIANCE LIFE SCIENCES PVT.LTD.
an Indian Company having its Registered office at
Chitrakoot, 2nd Floor,
Ganpatrao Kadam Marg,
Shree Ram Mills Compound,
Lower Parel, Mumbai 400 013,
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed: -
FIELD OF THE INVENTION
The present invention relates to a chimeric protein of recombinant human granulocyte colony stimulating factor comprising of hG-CSF fused to an affinity fusion tag at its amino terminus.
BACKGROUND OF THE INVENTION
Granulocyte colony stimulating factor (G-CSF) is a 20 kDa glycoprotein cytokine that stimulates growth and development of granulocyte progenitors in the bone marrow (Nagata et al., 1986, Nature (London) 319, 415-418). It is produced by monocytes, macrophages, fibroblasts, and endothelial cells. It stimulates the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils. The molecular cloning of GCSF gene (Nagata et al., 1986, Nature (London) 319, 415-418; Souza LM et al., 1986, Science 232, 61-64) led to the production of recombinant protein and large scale manufacturing of the product. It was approved for use in humans by FDA in 1991. Recombinant human G-CSF is a potent stimulant of granulopoiesis and is useful in preventing infectious complications of some neutropenic states. It is widely used to accelerate neutrophil recovery from myelosuppressive treatments. G-CSF decreases the morbidity of cancer chemotherapy by reducing the incidence of febrile neutropenia, the morbidity of high-dose chemotherapy supported by marrow transplantation, and the incidence and duration of infection in patients with severe chronic neutropenia. G-CSF can be used to increase the numbers of circulating haemopoietic precursor cells which are then harvested by leucapheresis and used either as
a supplement to or a substitute for aspirated bone marrow cells in autologous haemopoietic cell transplants.
The natural human glycoprotein exists in two forms of 174 and 177 amino acids. The more abundant and more active 174 amino acid form has been used in the development of pharmaceutical products by recombinant DNA technology. Though it is a glycoprotein, non-glycosylated GCSF protein expressed in E. Coli was also found to be biologically active (Wingfieldetal., 1988, Biochemical Journal, 256, 213-218).
The recombinant human G-CSF synthesised in an E. coli expression system is called filgrastim. European patent no. EP0237545, published on 1987-09-23 by Lawrence M Souza discloses the production of pluripotent granulocyte colony stimulating factor using E.coli as a host organism. Another form of recombinant human G-CSF called lenograstim is synthesised in Chinese hamster ovary (CHO) cells. European patent no. EP0169566 published on 1986-01-29 by Ono Masayoshi et al., describes a novel colony stimulating factor (CSF) that has the ability to promote the differentiation and proliferation of human bone marrow cells to neutrophils, and a method to produce the same from a novel cell line which has been established from tumor cells in patients with oral cancer,
The most published studies have used filgrastim as it was the first form of G-CSF to be approved. Moreover since high levels of biologically active protein expression are achieved in E. coli, it is the host of preference for producing G-CSF. Esherichia coli is a single-celled organism that reproduces mainly through asexual reproduction. The carbon source is simple, and it does not require elaborate facilities for growth and maintenance. The simplicity of an E.coli makes it easy and cheap to work with. Moreover its rapid
growth cycle allows for a quick increase in the population size of a particular strain. However G-CSF in E. coli is expressed as inclusion bodies in the form of insoluble protein, which necessitates the additional steps for the isolation and purification of GCSF. Subsequent solubilization and renaturation of G-CSF is a must to give correctly folded biologically active form, which is typically a cumbersome and inefficient process. Therefore it is desirable that the recombinant G-CSF should be expressed in a soluble form.
One way to produce recombinant human G-CSF in soluble form is to produce chimeric protein formed by expression of a hybrid gene made by splicing together two gene sequences, In this approach, ideally GCSF would be operationaffy finked to an affinity tag so as to give chimeric protein with better solubility. A short linker may be present between the affinity tag and GCSF such that it could be cleaved upon purification. However, this approach would fail should the activity of the protein be lost, due to steric hindrance resulting in loss of the three dimensional structure of the protein.
Thus it is desirable to produce chimeric protein of recombinant hG-CSF with better solubility while preserving its activity.
United states patent application no. 20030082679, published on May 1 2003 (Sun, Lee-Hwei K.; et al.) discloses Fc fusion protein of human granulocyte colony-stimulating factor with increased biological activities. A preferred host cell for expressing fusion protein is Chinese Hamster Ovary cell line deficient in the DHFR since Fc fragement is also glycosylated. The invention does not disclose the expression of rhG-CSF in E.Coli and is directed primarily towards improving the activity of the protein. It does not address
the issue related to providing a fusion protein of G-CSF in a soluble form or the expression of G-CSF at amino terminal fusion tag.
Therefore it is clear that the need continues to exist for providing chimeric protein of recombinant hG-CSF to improve the solubility for providing ease in purification thereby improving the process. Additionally, a functional hGCSF molecule with an affinity tag would be very useful for a variety of applications.
OBJECTS OF THE INVENTION
Accordingly the subject invention provides a chimeric protein of recombinant human granulocyte colony stimulating factor comprising of hG-CSF fused to an affinity fusion tag at amino terminus.
The subject invention also provides a chimeric protein of recombinant human granulocyte colony stimulating factor optionally comprising of a cleavable linker between hG-CSF and affinity fusion tag.
The subject invention also provides a chimeric protein of recombinant human granulocyte colony stimulating factor for improving solubility of hG-CSF in E.coli.
The subject invention also provides a chimeric protein of recombinant human granulocyte colony stimulating factor for improving solubility of hG-CSF which should also retain its desirable characteristics.
The subject invention also provides a chimeric protein of recombinant human granulocyte colony stimulating factor that can be useful in developing diagnostic assays,
antibodies against G-CSF and the like.
The subject invention also provides a chimeric protein of recombinant human granulocyte colony stimulating factor that can be useful for raising antibodies to G-CSF for use in diagnostics and therapy.
The subject invention also provides a chimeric protein of recombinant human granulocyte colony stimulating factor that can be useful for ex-vivo granulopoieses.
The subject invention also provides a chimeric protein of recomninant human granulocyte colony stimulating factor that can be useful for growth and propagation of stem cells.
The subject invention also provides a chimeric protein of recombinant human granulocyte colony stimulating factor that can be useful for identifying novel proteins or factors that interact with hG-CSF.
BRIEF DESCRIPTION OF FIGURES:
Fig 1. Schematic representation of cloning strategy for the contraction of chimeric protein of G-CSF with MBP.
Fig 2 - Sequence ID 1 of chimeric protein of G-CSF with MBP.
Figure3: Expression of MBP-GCSF fusion protein.
A protein band at approx 62 kDa consistent with the size of MBP-GCSF is induced only in pMALc2x-GCSF clones. A 42 kDa protein corresponding to the size of MBP protein is induced when vector alone is expressed (lanes 2 and 3)
Figure 4: Confirmation of MBP-GCSF expression
The 62 kDa protein present in the induced lysates (I) and not in un-induced lysates (U) is recognized both by anti-MBP polyclonal antibody (New England Biolabs) and anti-GCSF antibody (Santa Cruz Biotechnologies).
Figure 5: Solubility of MBP-GCSF.
Two independent clones were analyzed for solubility of recombinant protein. In each case, a substantial part of the fusion protein was found in the soluble fraction suggesting that the protein was fairly soluble.
The present invention provides for a chimeric protein of recombinant human granulocyte colony stimulating factor comprising of hG-CSF fused to affinity fusion tag at its amino terminus.
In accordance with the present invention the chimeric protein of recombinant human granulocyte colony stimulating factor optionally comprisirig of a linker between hG-CSF and affinity fusion tag. The linker may be cleavable by using proteases.
The affinity fusion tag that may be employed in this invention is Maltose binding
protein (MBP). MBP has a molecular mass of about 42 kilodaltons. It has been employed as a fusion partner as it generally improves the solubility of the expressed protein in bacteria and can made to either express in the cytoplasm or periplam of E. Coli (Bassford, P J., Jr., T. J. Silhavy, et al. (1979). "Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm." J Bacteriol 139(1): 19-31; Di Guan, C, P. Li, et al. (1988). "Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein." Gene 67(1): 21-30; Chen, G. Q. and J. E. Gouaux (1996). "Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin." Protein Sci 5(3): 456-67). Though MBP tag is generally employed to improve the solubility of the fusion protein, it fails to do so some of the time (New England Biolabs Tech Data Sheet, 1992; Smith and Johnson, 1988, Kapust, R. B. and Waugh, D. S. (1999), Escherichia coli maltose-binding protein in uncommonly effective at promoting the solubility of polypeptide to which it is fused Protein Sci. 8, 1668 -1674). However surprisingly in the present invention MBP is found to improve solubility of chimeric protein while retaining the functional activity of hG-CSF as well as of MBP.
Chimeric protein of recombinant human granulocyte colony stimulating factor comprising of hG-CSF fused to affinity fusion tag in the present invention can be
prepared by method comprising:
a. obtaining a cDNA encoding hG-CSF or fragment thereof;
b. operationally linking DNA encoding affinity fusion tag MBP to DNA
encoding for hG-CSF optionally with an intervening sequence encoding a
c. cloning into suitable expression vector the DNA encoding hG-CSF in frame
with DNA encoding maltose-binding protein;
d. transferring the expression vector as containing the above DNA encoding
hG-CSF in frame with DNA encoding maltose-binding protein in host cell;
e. culturing the cloned host cell under the conditions which allow the
expression of the chimeric protein;
f. purifying the chimeric protein;
g. confirming the identity of the chimeric protein by SDS-PAGE and Western
Human cDNA encoding G-CSF employed in the present invention for preparation of fusion protein may be either full length or a segment of G-CSF open reading frame. The said sequences may be obtained by standard recombinant DNA techniques. G-CSF encoding sequence for the present invention may be synthesized or isolated from cDNA of the mammalian cells or obtained by screening cDNA library, RNA, first strand DNA or any other appropriate source.
G-CSF DNA sequence was ligated in-frame with maltose-binding protein encoding region into expression vector using appropriate methods known in the art like by employing restriction enzymes and ligases.
Promoters or enhancers employed in the present invention to affect and regulate the expression of chimeric protein can be selected from prokaryotic or eukaryotic promoters. The prokaryotic promoters that may be used in the present invention can be selected from LAC promoter, TAC promoter, beta-lactamase promoter or the like, The eukaryotic promoters that may be used in the present invention can be selected from CMV immediate early promoter, SV 40 early or late promoter, retroviral Long Terminal Repeat (LTR), Polyhedirn promoter, EF-1, beta-actin promoter or the like.
The chimeric protein of the present invention comprising of hG-CSF fused to affinity fusion tag at the carboxy terminus was subjected to functional assays to check the biological activities.
The chimeric protein of the present invention comprising G-CSF fused to affinity fusion tag-maltose binding protein apart from improving the solubility of the protein to provide the ease in purification can also be used for various other applications like the chimeric protein can be used in diagnostic assays for G-CSF, ex-vivo proliferation or expansion of granulocytes, for raising antibodies to G-CSF for
use in diagnostics and therapy, for identifying novel proteins or factors that interact with G-CSF and for ex-vivo growth and development of stem cells.
The various features and aspects of the present invention are illustrated with the help of following non-limiting examples.
Cloning of GCSF into pMAL vector:
Plasmid vectors carrying MBP coding sequence (pMAL-c2x and pMAL-p2x, US Patent 5643758 from New England Biolabs, Beverly, MA) were cut with XmnI restriction endonuclease enzyme and used for ligating GCSF coding region inframe with MBP. The GCSF cDNA was amplified from RNA extracted from U-87MG cell line through standard procedures. The published GCSF sequence (Genbank Acc# NM_000759) was used to design a set of primers (Forward - 5' CAT ATG ACC CCC CTG GGC CCT GCC - 3' and Reverse primer 5'- GGA TCC TCA GGG CTG GGC AAG GTG -3') to amplify the GCSF ORF without the putative signal sequence coding region. The primers also allowed the addition of an Ndel site at the 5' end and a BamHI site at the 3' end. The PCR product was ligated using T4 DNA ligase into pMALc-2x and pMAL-p2x cut with XmnI (Fig. 1). Restriction analysis and sequencing by dideoxy chain termination method confirmed that the GCSF sequence was in-frame with MBP coding sequence (Fig. 2).
Expression of MBP-GCSF fusion protein:
E.coli (TB-1, New England Biolabs) carrying MBP-GCSF fusion constructs (pMAL-p2x-GCSF or pMAL-c2x-GCSF) and vector alone were each grown overnight in a sterile 5 ml LB (lOg Tryptone, 5 g Yeast Extract and 5g Nacl) containing ampicillin (100 ug/ml). The overnight culture was used to set up a 5 ml culture (1:30 dilution) and grown for an additional 2 hours (OD6oo of 0-5) and induced with IPTG (0.3 mM). 1 ml aliquots were taken at 2.5 hours after induction, pelleted by centrifugation and lysed in a buffer containing lysozyme (200 ug/ml). The lysates were separated on 10% SDS-PAGE and stained with commassie stain. An induced protein band corresponding to MBP-GCSF (approx 62 kDa) was evident (Fig. 3) suggesting mat the fusion protein was indeed produced in E. coli.
To confirm that the protein was MBP-GCSF, the lysate was resolved on SDS-PAGE and transferred to Nitrocellulose membrane and probed with anti-MBP polyclonal antibody (New England Biolabs, Beverly, MA) and anti-GCSF anti-peptide antibody (Santa Cruz Biotechnology, CA). (Fig .4). As evident from the blot, the 62 kDa band was recognized by both anti-MBP and anti-GCSF antibodies confirming that the protein band is indeed the fusion protein.
Fusion with MBP enables high level of GCSF expression:
To test if the level of expression is enhanced by the presence of MBP tag, we removed the MBP coding region by digesting pMALc2x-GCSF with Ndel enzyme. This ensures that GCSF start site is intact and replaces the MBP start site in the plasmid. The plasmids
with MBP coding region deleted were transformed into TB-1 cells and tested for GCSF expression. No significant induced band of approx 20 kDa was visible on a commassie stained gel. The sequence was confirmed through dideoxy sequencing and no mutations were observed in the coding region. This may be codons at the 5' end that are not preferred by E. coli (Jeong, K. J and Lee, S.Y. (2001) Secretory production of human granulocyte colony stimulating factor in E. coli. Protein Expression and Purification. 23(2):311-318)
Fusion tag improves solubility of GCSF:
GCSF is highly insoluble when expressed in E. coli (Souza, L. M., at al., (1986) Recombinant human granulocyte colony stimulating factor: effects on normal and leukemic myeloid cells, Science, 232: 61-65). To test if MBP-GCSF is expressed as a soluble or insoluble protein, E. coli expressing MBP-GCSF were lysed gently by sonication in PBS and resolved on a 10% SDS-PAGE gel. The protein bands were visualized upon staining with coomassie blue as earlier. A significant portion of MBP-GCSF was present in the soluble fraction consistent with enhanced solubility of the fusion protein (Fig. 5).
Fusion tag allows easy purification of GCSF for immunizations and hybridoma development:
Since the fusion protein contains full length GCSF with MBP tag, amylose resin affinity
chromatography can be used to purify the protein using established procedures (New England Biolabs, pMAL protein Fusion and Purification System, Instruction manual, p23). Briefly, one liter of cells expressing the fusion protein were harvested by centrifugation at 4000 g for 20 minutes and the supernatant was discarded. The cells were resuspended in 50 ml of Column Buffer (20 ml of 1 M Tris-HCl, pH 7.4, 11.7 g of NaCl, 2.0 ml of 0.5 M EDTA per liter containing 154 mg of DTT). The sample was frozen at-70 degrees overnight and thawed in cold water. The sample was sonicated and the soluble fraction was collected by centrifuging at 9000g for 30 minutes. The supernatant was diluted five-fold in column buffer. Amylose resin (New England Biolabs) was used to purify the protein according to manufacturer's instructions. The purified proteins were resolved on SDS-PAGE and stained with commassie stain to assess the purity. No refolding was necessary since the protein was soluble and did not have to be denatured prior to purification. It can be used to immunize balb/c mice to produce monoclonal antibodies both against the tag and the protein.
1. A chimeric protein of G-CSF with affinity fusion tag maltose binding protein having SEQUENCE ID 1.
2. The chimeric protein as claimed in claim 1, wherein the affinity fusion tag is present at the N- terminus of G-CSF.
3. The chimeric protein as claimed in claim 1, wherein the affinity fusion tag is present at the C- terminus of G-CSF.
4. The chimeric protein as claimed in claim 1, wherein the affinity fusion tag is fused to G-CSF complete sequence.
5. The chimeric protein as claimed in claim 1, wherein the affinity fusion tag is fused to part of G-CSF sequence.
6. The chimeric protein as claimed in claim 1, wherein the affinity fusion tag improves solubility of G-CSF.
7. The chimeric protein as claimed in claim 1, wherein the affinity fusion tag improves expression level of G-CSF.
8. Chimeric protein of recombinant human granulocyte colony stimulating factor
comprising of hG-CSF fused to affinity fusion tag in the present invention can
be prepared by method comprising: h. obtaining a cDNA encoding hG-CSF or fragment thereof; i. operationally linking DNA encoding affinity fusion tag MBP to DNA
encoding for hG-CSF optionally with an intervening sequence encoding a
linker j. cloning into suitable expression vector the DNA encoding hG-CSF in frame
with DNA encoding maltose-binding protein; k. transferring the expression vector as containing the above DNA encoding
hG-CSF in frame with DNA encoding maltose-binding protein in host cell;
1. culturing the cloned host cell under the conditions which allow the expression of the chimeric protein; m. purifying the chimeric protein; n. confirming the identity of the chimeric protein by SDS-PAGE and Western blot.
14. A recombinant fusion protein as claimed in claim 1 to 8 and illustrated with examples 1 to 5
Dated this 31st day of March 2005.
For RELIANCE LIFE SCIENCES PVT.LTD. .
Sr. Executive Vice President
The Controller of Patents The Patent Office Mumbai.
|Indian Patent Application Number||395/MUM/2004|
|PG Journal Number||24/2010|
|Date of Filing||31-Mar-2004|
|Name of Patentee||RELIANCE LIFE SCIENCES PRIVATE LIMITED|
|Applicant Address||CHITRAKOOT,2ND FLOOR,GANPATRAO KADAM MARG,SHREE RAM MILLS COMPOUND,LOWER PAREL,MUMBAI 400 013,|
|PCT International Classification Number||A61K39/00|
|PCT International Application Number||N/A|
|PCT International Filing date|