|Title of Invention||
ONE STEP FLOW-THROUGH ADSORPTIVE PURIFICATION OF TUBULIN FROM TISSUE HOMOGENATE
|Abstract||Microtubules that are essential for a variety of cellular functions prove to be the targets of anticancer drugs that can induce apoptosis in cells. This poses the need for increasing quantities of pure microtubule preparations that can retain their biological activity for in vitro experiments, a and P tubulin along with associated proteins that form the major constituent of microtubules are classically purified by tedious cycles of polymerization-depolymerization yielding tubulin preparations with high contamination of microtubule associated proteins. Attempts to removing the microtubule-associated proteins have been associated with the loss of tubulin. Here we report the process of separation of tubulin dimers from mammalian brain extract by use of a single step purification method. Tubulin polymers along with associated proteins are separated from the other soluble cell components by use of "Negative Chromatography". The tubulin polymers flow through a packed bed of macroporous chromatographic media with inherent hydrophobic and a combination of hydrophobic and ionic character that adsorbs all the other cell components. The flow-through mass obtained is further purified by a depolymerization step that yields tubulin dimers which exhibits biological activity detected by the capacity of the microtubule to reassociate under favorable conditions and also exhibit colchicine-mediated inhibition of the purified tubulin.|
|Full Text||COMPLETE AFTER PROVISIONS LEFT ON
" 5 MAY 2005
THE PATENTS ACT, 1970
(39 of 1970)
THE PATENT RULES 2003
(See Section 10 and rule 13)
1. One Step Flow through Adsorptive Purification of Tubulin from Tissue Homogenate.
2. (a) Lali Arvind Mallinath.
Chemical Engineering Division
University Institute of Chemical Technology,
Nathalal Parikh Marg,
The following specification describes the nature of this invention
PREAMBLE TO DESCRIPTION : COMPLETE
Field of Invention
The present invention relates to a single step process for purification of polymers of tubulin from mammalian brain tissue homogenate employing a macroporous chromatographic media with an inherent hydrophobic and a combination of hydrophobic and ion exchange character. This novel process, whereby 'polymerized' tubulin is obtained as unadsorbed fraction while passing through a column packed with the macroporous media, provides purified tubulin free from other soluble constituents of the tissue homogenate, and is obtained in its bioactive form. The tubulin obtained by the present invention has various biological applications and can be used as a target for designing therapeutic drugs in cancer research.
Background of the Invention (Prior Art):
Microtubules are polymers of dimeric subunits of a and (3 tubulin in dynamic equilibrium with the intracellular pool of tubulin dimers. They are the integral components of the mitotic spindle and are involved in non-mitotic functions such as chemotaxis, membrane and intracellular scaffolding, neurotransmission, anchorage of sub-cellular organelles to the receptors, cell adhesion and locomotion and transmission of cell signaling. The dynamic reaction of equilibrium towards polymerization and depolymerization, crucial for the structural integrity of microtubules is influenced by several co factors, including guanosine 5'-triphosphate (GTP), the ionic environment and microtubule-associated proteins. Antimicrotubule agents viz., vinca alkaloids, taxanes and an increasing number of both natural products and synthetic compounds, can disrupt this dynamic equilibrium of microtubules and therefore tubulin preparations find wide applications in the field of designing targets that can be used as drugs for future cancer therapy.
Increase in instances of cancer has led us to an extensive search for antimitotic compounds, natural as well as synthetic compounds. The compounds need to be assessed for their potential to act as tubulin binding agents (TBAs). Thus, tubulin plays a central role in the studies and development of growing number of anticancer drugs during the last decade. Commercial tubulin
preparations while available at high prices are unstable often necessitating indigenous preparation as and when required. Thus the cost and the short shelf life of tubulin preparations have been a hurdle in the direction of the above research. Therefore, constant efforts are on to simplify and develop a cost effective process for the purification of tubulin from mammalian tissue that cannot facilitate large-scale production but also speed up biomedical research involving tubulin.
The driving force behind the purification of tubulin has been the concept that "Tubulin is the only protein, which polymerizes at 37°C and depolymerises at 4°C". Exploiting the principle, various strategies have been reported in the literature that follow extensive cycles of polymerization and depolymerization to purify tubulin.
Shelanski et.al (1973) reported the separation of guinea pig tubulin by two cycles of assembly in glycerol/sucrose containing solutions in the absence and presence of ATP or GTP. The procedure had the advantages of selecting only those subunits capable of polymerization and, presumably, eliminating the presence of denatured protein in the preparation. The extraordinary stability of the normally labile tubulin subunits when stored in glycerol was also highlighted.
Lee et.al (1973) reported the purification of tubulin by DEAE-Sephadex anion-exchange adsorption chromatography followed by ammonium sulfate precipitation. The preparation was then desalted using Sephadex-G25 coarse, and the preparation was repolymerized employing MgCl2.
Murphy et.al (1975) separated tubulin from the high-molecular-weight components (HMW) specifically associated with microtubule proteins by DEAE-Sephadex anion exchange chromatography. The starting material for the chromatographic process was the cold-depolymerized microtubule preparations obtained from repeated cycles of polymerization-depolymerization.
Berkowitz et.al (1977) reported the use of gel-filtration chromatography of cold-depolymerized microtubule preparations to separate a polydisperse fraction of high molecular weight containing non-tubulin proteins from tubulin.
Hamel et.al (1981) presented a three-step method for the large-scale purification of calf brain tubulin exploiting the ability of high concentration of glutamate to stabilize tubulin and to induce its polymerization in the presence of GTP.
Roychowdhury et.al (1984) employed fast-performance liquid chromatography using Mono Q column (anion exchanger) to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly-disassembly. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components that were removed by adsorption on Mono S (cation exchanger) columns where tubulin was isolated as the flow-through fraction. Therefore, a combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified tubulin protein.
Pal M et.al (1990) have reported the purification of tubulin protein from Mimosa pudica fresh leaves and pulvinar callus cells, using an anion-exchange resin, DEAE-Sephadex A-50, followed by ammonium sulfate fractionation and Sephadex G-200 gel filtration.
Edelstein SJ et.al (1993) developed a two-step procedure for the obtaining 90% pure tubulin from Sacchromyces cerevisiae. The high-speed supernatant of lysed cells was loaded onto a DEAE-Sephadex column followed by the tubulin fractions being loaded onto an inimunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to SepharoseB. The preparation however lacked the ability to polymerize by itself and required the addition of porcine brain microtubule-associated proteins or DEAE-dextran for assembly into microtubules.
Oakley BR et.al (1995) demonstrated the overproduction of tubulin in Aspergillus nidulans with partial purification by ion-exchange chromatography, and final purification by rounds of assembly and disassembly.
Sackett DL (1995) reported the purification of tubulin from less than 100 mg of tissue or tissue culture cell protein in less than 6 h using a combination of a solid-phase ion exchanger (MemSep DEAE) and one cycle of temperature-dependent polymerization and depolymerization.
Sloboda RD et.al (1998) demonstrated that membrane ion exchangers mounted in syringe filter cartridges can be used to separate tubulin from MAPs in cold-depolymerized preparations. The resulting tubulin was competent to assemble into microtubules upon either addition of the purified MAPs or addition of the microtubule-stabilizing drug Taxol.
Werbovetz KA et.al (1999) reported the purification of tubulin from kinetoplastid parasites that may be used as an excellent drug target. The tubulin was purified on a milligram scale from Leishmania mexicana amazonensis promastigotes by sonication, DEAE-Sepharose chromatography, and one cycle of assembly-disassembly. The purified leishmanial tubulin was recognized by commercially available anti-tubulin antibodies and displayed concentration dependent assembly in vitro.
Banerjee (1999) reported the use of an immunoaffinity column whereby bovine brain tubulin was fractionated into three functionally active alpha beta heterodimers that were identified by immunoblotting with alpha-tubulin-specific antibodies and sequence analysis. Assembly studies in the presence of glycerol and Mg2+ demonstrated the presence of tyrosinated form of alpha tubulin, that assembled poorly, and the non-tyrosinated form that assembled normally.
Castoldi M et.al (2003) reported that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allows the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol was rapid and was reported to yield more active tubulin than the traditional ion-exchange chromatography-based procedures.
All of the above reported procedures employ strategies for separating tubulin from the cold-depolymerized microtubule preparation. The disadvantages of using the alternating cycles of polymerization-depolymerization include loss of viable tubulin in each cycle, besides being greatly laborious and time consuming. Use of cold-depolymerized microtubule preparation necessitates the maintenance of low temperatures throughout all the steps of isolation. Moreover, the sulfonate buffers usually used for the different methodologies are expensive making the final cost of the method exorbitant to be carried out on large scale. There are also
reports stating that use of ion-exchange chromatography for the separation of tubulin from cold-depolymerized microtubule preparation usually causes the loss of bioactivity of the final product making the whole process unfit for use.
The reported protocols involve use of mammalian brain (sheep/goat/pig/calf) or parasites (Leshmania I Trypanosoma) or yeast (Sacchromyces cerevisiae) as the source of tubulin present to the tune of 8 to 10% of the soluble protein. The traditional method, which is followed by researchers worldwide, employ three cycles of temperature induced polymerizations and depolymerizations. The entire isolation procedure is carried out in cold (4°C) except the polymerization step carried out between 25-40°C.
Use of chemical agents that can induce tubulin polymerization has also been reported. These agents are intended to give better yields of more stable tubulin preparations. Thus, agents like glycerol, monosodium glutamate, DEAE-dextran, dimethylsulphoxide, sucrose, polyethylene glycol, and high concentrations of Mg have been used.
In all procedures reported and used, each polymerization step is followed by high-speed ultracentrifugation that separates the polymerized tubulin from other soluble proteins. Similarly, each depolymerization step is also followed by high-speed ultracentrifugation to remove any undissolved components. Electrophoresis of microtubule preparations purified by repeated cycles of assembly and disassembly has shown that they still contain many proteins (Microtubule-Associated Proteins-MAPS) in addition to alpha- and beta-tubulin., and that these proteins constitute about 17% of the total material present.
The references discussed herein are provided solely for their disclosure prior to filing date of present application. Nothing herein to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
Objective of Invention
The primary objective of this invention is to propose a new and simple process for separation and purification of bioactive tubulin with high purity from mammalian brain tissue homogenate. The above objective has been achieved by employing a simple, economical, and commercially viable
process using "Negative Chromatography" whereby the polymerized tubulin is allowed to pass through the column bed of a macroporous adsorbent that is able to bind all other components of the homogenate through hydrophobic or combination of hydrophobic and ionic interactions.
All reported and used procedures exploit extensive cycles of polymerization and depolymerization to purify tubulin making the purification laborious and expensive. One of the main objectives of the invention was to reduce the time consumed, and use a single step chromatographic method for efficient separation. Attempts at purification on column chromatography, have usually exploited low temperatures as the tubulin dimer is denatured at high temperatures, thereby also highlighting the need for a purification method that can be employed at room temperatures. The additional advantage of working at near room temperatures can be greater stability of polymerized tubulin, and hence higher overall yield of active tubulin.
Repeated rounds of temperature changes in polymerization-depolymerization steps are known to cause loss in the final yields of the product. Use of single step of polymerization-depolymerization with an intermediate "Negative Purification" step using our invention results in increase of the final yield of tubulin, and simplifies the entire procedure to a large extent. The dilution of the sample that is a result of any chromatographic step is also minimized as the polymerized mass is pelleted before it is reconstituted in the depolymerization buffer.
Another new aspect of the present invention is the use of buffers other than reported and used so far. Sulfonate based buffers for the polymerization and depolymerization of tubulin are accepted as buffers of choice for microtubule polymerization. However, use of these buffers increases the cost, and need for alternatives led us to explore use of more common buffers e.g. Tris based buffer, and phosphate based buffers, in order to reduce the cost of the process.
The present process is therefore a single step convenient, cost effective and scalable method for isolation of tubulin from the polymerized tubulin-tissue homogenate preparation without loss of biological activity.
Brief Description of the Drawings
Figure 1: Schematic view of preferred embodiment of the process according to the invention wherein the polymerized mammalian brain tissue homogenate is processed. 1: Column 1 (packed bed), 2: Column 1 adaptor, 3: UV-spectrophotometer, 4: Chart recorder, 5: Peristaltic pump, 6:Ultra centrifuge, SI: Equilibration Buffer, S2: Polymerized tubulin-tissue homogenate maintained in a water bath at the required temperature, S3: Washing Buffer, S4: D. M. Water, S5: Regenerant Solution, S6: D. M. Water, Fl: Flow-through turbid fractions, F2: Proteins bound to the resin that are removed when regeneration buffer is passed through the column, F3: Depolymerized tubulin preparation that is processed from the flow-through fractions after centrifugation and redissolving the pellet. Tanks SI to S6 are connected in series with care as that there is no mixing of their contents with each other. Direction of cycle is from SI to S6.
Figure 2: Elution profiles for the flow-through fractions demonstrating total protein content as mg (Bradford assay) as function of fractions collected (1 through 11, each 50mL) for a typical purification experiment of passing goat brain tissue homogenate through a 25mm diameter glass column packed with the adsorbent hydrophobic CELBEADS to a settled bed height of 100mm, and charged with 80mL of polymerized tissue homogenate at a superficial linear flow velocity of lOOcm/hr. The two profiles shown are for runs made in - A: glycerol as the polymerization inducing agent; and B: glutamate as the polymerization inducing agent.
Figure 3: Elution profiles for the flow-through fractions as turbidity content of the eluted fractions (1 to 12, each 50mL) for a typical purification experiment of passing goat brain tissue homogenate through a 25mm diameter glass column packed with the adsorbent hydrophobic CELBEADS to a settled bed height of 100mm, and charged with 80mL of polymerized tissue homogenate at a superficial linear flow velocity of lOOcm/hr. The two profiles shown are for runs made in - A: glycerol as the polymerization inducing agent; and B: glutamate as the polymerization inducing agent.
Figure 4: Show 12% SDS-PAGE of purified tubulin from mammalian tissue homogenate. Sample 1: Tissue homogenate, Sample 2: Purified tubulin sample, Sample 3: Low molecular weight markers.
Description of Present Invention
In the present invention, the polymerized tubulin containing tissue homogenate obtained from the homogenized and cold centrifuged supernatant, is directly passed through a column packed with a macroporous adsorbent matrix, whereby the polymerized tubulin is able to pass through un-adsorbed, while all other soluble components of the homogenate bind to the macroporous matrix through hydrophobic, or combination of hydrophobic and ionic interactions, resulting in the eluting fractions containing purified bioactive polymerized tubulin.
The present invention focuses on the use of polymerized-tubulin tissue homogenate prepared by the homogenized goat brain tissue in the presence of as tubulin polymerization inducing agents. The tissue homogenization is carried out in buffers (pH 6-8) so that the integrity of the tubulin polymer is maintained and there is no loss of bioactivity. Higher ionic concentrations of buffers can be used for better yields. The polymerization is carried out in the presence of GTP (sodium salt of Guanosine Tri Phosphate) required for maintaining the dynamic equilibrium of the tubulin polymers and EGTA (Ethylene glycol-0, 0'- bis (2-aminoethyl) N, N, N', N'- tetraacetic acid) which functions as a chelating agent to trap calcium ions that may act as the inhibitor for tubulin polymerization. The tubulin in the tissue homogenate is capable of polymerizing when incubated at temperatures between 25-37°C making the solution homogenously turbid. This turbid mass functions, in this invention, as the feed for purification of tubulin. To reduce the chances of clogging in the column care has to be taken so that the polymerization step does not produce settling agglomerate mass.
The polymerized tubulin-tissue homogenate is processed by chromatography in an attempt to purify the polymerized tubulin from other contaminating proteins. The selection of the resin for the purpose of purification was made with the following two aspects in mind: 1) the pores of the resin should be large enough so that the tubulin aggregates/polymers can efficiently pass through them without clogging the bed, pores and being entrapped; and 2) the resin should possess sufficient hydrophobic and/or ionic characteristics to bind all other proteins in the brain tissue homogenate. Based on the above requirements, hydrophobic CELBEADS (an indigenous resin preparation - Indian patent application No. 356/MUM/2003), DIAION® HP-20 (Resindion Sri,
Mitsubishi Chemical Corp. Binasco, Italy) and SEPABEADS® EB-DA (Resindion Sri, Mitsubishi Chemical Corp. Binasco, Italy) series were used. CELBEADS series and adsorbent resins belonging to the DIAION® series are hydrophobic, while resins belonging to the SEPABEADS® series are ion-exchange matrices with a partial hydrophobic character. These matrices possess the macropores i.e. pore size anywhere in the range 300 to 30,000um, and these play a crucial role in the flow-through of polymerized tubulin, and capture of contaminants.
In the preferred embodiment, the column is charged with a rigid, macroporous hydrophobic or a combination of hydrophobic and ionic interactions resins. The adsorbent is then equilibrated with buffered aqueous solution having pH in the range of 6.0-8.0, having ionic strength of 70 to 700mM, with polymerization inducing agent selected from but not limited to glutamate and glycerol, in presence of EGTA, GTP and ATP. The buffer can be selected from but not limited to sulfonate buffers, phosphate buffer, acetate buffer, and Tris buffer. Equilibration can be done in packed bed with 3 to 4 bed volumes of the equilibrating buffer. After required equilibration of the column, the polymerized tubulin-tissue homogenate is charged at the desired flow velocity from 50cm/h to 200cm/h. After loading the homogenate, the column is washed with equilibrating solution, possibly at the same flow velocity as that during loading. The polymerized tubulin fractions flow through the column as turbid unbound proteins while the other soluble proteins are adsorbed to the resin.
The pooled flow-through fractions are further processed by centrifugation wherein the polymerized tubulin settles down as a pellet while the residual contaminating proteins are separated in the supernatant. The pellet i.e. the polymerized tubulin is solubilised in a buffered aqueous solution having pH in the range of 6.0-8.0, having ionic strength of 10 to lOOmM, in presence of EGTA, GTP and ATP. The buffer can be selected from but not limited to sulfonate buffers, phosphate buffer, acetate buffer, Tris buffer. The solubilised protein is then incubated at low temperatures for not less than 2 hrs following which it is subjected to centrifugation. The depolymerized and purified tubulin can be obtained as a clear supernatant. The adsorbent in the column can be used multiple times by employing a suitable cleaning-in-place procedure. Cleaning in place (CIP) of the column is carried out applying by 4 to 5 bed volumes of 0.5 to 1.0M NaOH followed by D. M. water wash till pH falls to neutral. This makes
the column ready for the next cycle of equilibration, loading, washing, and elution in the specified way for the optimal recovery of tubulin from brain tissue homogenate.
Quality control tests for tubulin
(1) Purity of tubulin is determined by applying samples of at least lOjag of protein to SDS polyacrylamide (12% w/v) slab gel electrophoresis (SDS PAGE). Protein bands are visualized by staining with silver stain.
(2) Bioactivity of tubulin is determined by turbidometric analysis at 350nm. ImL of the preparation is allowed to polymerize in the presence of ImM GTP, 33% DMSO and 0.33 M monosodium glutamate. The viable samples produce a consistent increase in absorbance for a period of 10 minutes measured at 350nm on a spectrometer. The capacity of the inhibitor molecule "colchicine" to inhibit the polymerization is also assessed. This assay involves addition of lOOul of ImM of colchicine to ImL of the sample and allowed to polymerize in the presence of ImM GTP, 33% DMSO and 0.33M monosodium glutamate. The reduction/inhibition of the polymerization observed for 10 minutes is an indicator of the bioactivity of the purified preparation.
NOVEL FEATURES OF THE INVENTION:
The present invention focuses on the separation of polymerized tubulin from the tissue
homogenate. The novelty of the invention lies in
1. The use of the polymerized tubulin tissue homogenate for loading the column packed with a macroporous adsorbent that is capable of separating the tubulin polymers from the soluble proteins in the homogenate. This reduces the total time required for the separation of tubulin compared to the traditional methods of purification making the process fast and less tedious. The temperatures required for handling the polymerized mass are near to room temperature making the invention easy to handle. Moreover, the purified polymerized tubulin can be stored until use in its polymerized state and depolymerized as and when required.
2. Use of macroporous adsorbent so that the tubulin polymers can pass through the large pores of the adsorbent and flow through the bed of the resin, while the soluble constituents like proteins are either trapped in the resin or are adsorbed through
hydrophobic or combination of hydrophobic and ionic interactions on the resin. This strategy aids in a better, faster and easier separation of the tubulin protein from the mammalian brain tissue homogenate. 3. The improved yield of tubulin both in quality and quantity. The tubulin yields are comparable with maximum reported in literature, and the purified tubulin shows as a single band on SDS polyacrylamide (12% w/v) slab gel electrophoresis (SDS PAGE). The bioactivity of the depolymerized tubulin as assessed by the polymerization and its ability to bind to colchicines proved the fact that the bioactivity of the tubulin is preserved.
Example: 1: POLYMERIZATION USING 4M GLYCEROL ON CELBEADS
Load Preparation: Freshly cut goat brain is obtained from the local slaughterhouse. The brain tissue is cleansed of blood vessels and meninges. After being weighed, it is homogenized in 80mM Tris-buffer pH=6.8 containing 3mM MgS04, lmM EGTA (Ethylene glycol-0, 0'- bis(2-aminoethyl) N, N, N\ N'- tetraacetic acid) with the help of grinder under cold conditions. The homogenate is then centrifuged at 12000rpm at 4°C for half a hour30 minutes. The supernatant so obtained is mixed with the selected polymerizing agent of 4M glycerol and incubated at 25-37°C in the presence of lmM GTP for 1 hour. The resultant homogenous mass is then used as the load for the purification of tubulin from tissue homogenate.
Column parameters: A 25 mm inner diameter and 250mm long borosilicate glass column is filled with 75ml of CELBEADS. The settled bed height is 100mm and the flow-rate was set at lOOcm/hr. The matrix is equilibrated with 2.5 bed volumes of aqueous equilibrating solution containing 80mM Tris-buffer pH=6.8 containing 3mM MgSCM, lmM EGTA (Ethylene glycol-0, 0'- bis (2-aminoethyl) N, N, N', N'- tetraacetic acid) with the polymerizing agent (4M glycerol). To this pre-equilibrated column, 80 ml of the above load solution is passed at linear velocity of 100 cm/hr in a packed bed mode. The resin column is washed with the aqueous equilibrating solution containing 80mM Tris-buffer pH=6.8 containing 3mM MgSC^, lmM EGTA (Ethylene glycol-0,0'-bis(2-aminoethyl) N, N, N', N'- tetraacetic acid) with the polymerizing agent 4M glycerol till the protein concentration of the fractions was >0.1mg. The initial fractions exhibit turbidity along with high protein content. These fractions are then centrifuged at 12000 rpm at 27°C for a hour. The resulting pellet consists mainly of polymerized tubulin that is depolymerized after addition of 25mM Tris-buffer pH = 6.8 containing 3mM MgS04, lmM EGTA ( Ethylene glycol-0,0'- bis(2-aminoethyl) N, N, N\ N'- tetraacetic acid) and O.lmM GTP in cold conditions for 1 -2hrs.
The purity of the sample is determined by applying samples of at least 10 ug of protein to SDS-polyacrylamide (12% w/v) slab gel electrophoresis (SDS PAGE). Protein bands are visualized by staining with silver stain. The purified sample of tubulin using the above invention yielded a single protein band at 50kD. The tubulin preparation is further assayed for bioactivity that was determined by turbidimetric analysis at 350nm where lmL of the preparation is polymerized in the presence of lmM GTP, 33% DMSO and 0.33 M monosodium glutamate. The purified samples showed a consistent increase in absorbance for a period of 10 minutes. The turbidimetric
assay is shown to be inhibited by addition of lOOul of ImM of colchicine to ImL of the preparation and allowed to polymerize in the presence of ImM GTP, 33% DMSO and 0.33 M monosodium glutamate.
Example: 2: POLYMERIZATION USING IM GLUTAMATE ON CELBEADS
Load preparation: The tubulin homogenous mass was prepared as per the protocol in Example 1. The polymerizing agent used for the preparation was IM glutamate with an incubation of 45 mins at room temperature.
The CELBEADS column was packed as in Example 1 with all parameters constant.
The separation obtained under these conditions was identical to Example 1.
Example: 3: SEPARATION ON ION-EXCHANGE MATRIX Sepabeads EB-DA
Load preparation: The tubulin homogenous mass was prepared as per the protocol in Example 1. The polymerizing agent used for the preparation was IM glutamate with an incubation of 45 mins. at room temperature.
The Sepabeads EB-DA column was packed as in Example 1 with all parameters constant. The separation obtained under these conditions was identical to Example 1.
1. A new process for separation/purification of polymerized tubulin from tubulin containing
tissue homogenate obtained from the homogenized and cold centrifuged supernatant, by direct passage of the polymerized tubulin suspension through a column packed with a macroporous adsorbent matrix, whereby the polymerized tubulin is able to pass through un-adsorbed, while all other soluble components of the homogenate bind to the macroporous matrix through hydrophobic and/or ionic interactions, resulting in the eluting fractions containing purified bioactive polymerized tubulin. The process comprises of the following:
a. The tissue homogenate is incubated at near room temperatures, 25° to 40°C, so as to
facilitate the polymerization of tubulin in presence of one or many polymerization
inducers from GTP, glycerol and/or glutamate salts.
b. The homogenate prepared in any suitable buffer of molarity 50-100mM at pH between 6
to 8, and containing the polymerized mass, is passed through the bed of the macroporous
adsorbent at room temperature, or between 25° and 40°C, so that the polymerized tubulin
is obtained as flow through fraction.
c. The macroporous adsorbents used possess large enough pores, preferably with mean pore
size larger than 1000A, so that the polymers of tubulin flow through as un-adsorbed
fraction while the other soluble constituents are adsorbed on the matrix, thereby
separation of tubulin polymers is successfully carried out by "Negative
d. The adsorbing capacity of the macroporous adsorbent is such that its inherent
hydrophobic, or a combination of hydrophobic and ionic interactions with the soluble
proteins in the tissue homogenate are such that the polymerized tubulin passes
unadsorbed while all other soluble components are retained adsorbed.
e. The resulting tubulin preparation is able to give 20 to 30mg purified tubulin from lOOgm
of brain, or any other tubulin rich source, such that the purified tubulin is bioactive and is
homogeneous on SDS-PAGE.
ABSTRACT OF THE INVENTION
Microtubules that are essential for a variety of cellular functions prove to be the targets of anticancer drugs that can induce apoptosis in cells. This poses the need for increasing quantities of pure microtubule preparations that can retain their biological activity for in vitro experiments, a and P tubulin along with associated proteins that form the major constituent of microtubules are classically purified by tedious cycles of polymerization-depolymerization yielding tubulin preparations with high contamination of microtubule associated proteins. Attempts to removing the microtubule-associated proteins have been associated with the loss of tubulin. Here we report the process of separation of tubulin dimers from mammalian brain extract by use of a single step purification method. Tubulin polymers along with associated proteins are separated from the other soluble cell components by use of "Negative Chromatography". The tubulin polymers flow through a packed bed of macroporous chromatographic media with inherent hydrophobic and a combination of hydrophobic and ionic character that adsorbs all the other cell components. The flow-through mass obtained is further purified by a depolymerization step that yields tubulin dimers which exhibits biological activity detected by the capacity of the microtubule to reassociate under favorable conditions and also exhibit colchicine-mediated inhibition of the purified tubulin.
|Indian Patent Application Number||503/MUM/2004|
|PG Journal Number||10/2009|
|Date of Filing||05-May-2004|
|Name of Patentee||LALI ARVIND MALLINATH|
|Applicant Address||CHEMICAL ENGINEERING DIVISION UNIVERSITY INSTITUTE OF CHEMICAL TECHNOLOGY,NATHALAL PARIKH MARG,MATUNGA (E),MUMBAI-400 019,|
|PCT International Classification Number||A01N43/04|
|PCT International Application Number||N/A|
|PCT International Filing date|