THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
Section 10

"Extraction Of Recombinant Proteins From Inclusion Bodies Using A Denaturant Solution Containing An Organic Solvent"

Unichem Laboratories Ltd. an Indian company, of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

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Field of invention:
The instant invention relates to a process of extracting recombinant protein from inclusion bodies obtained from the E.coli.
Background of the invention:
In E.coli the over expressed protein often accumulates intracellularly in an insoluble form resulting in inclusions in the cytoplasm. The solubilization is the subject matter of this patent.
The majority of proteins present in the inclusion bodies is not in their native state and requires to be folded before they can exhibit their biological activity. Since these proteins are in their denatured state the proteins are first solubilized and purified before they are folded. Detergents such as cationic, anionic and nonionic have been used for the solubilization of proteins from inclusion bodies. Also solvents such as butanol have been used earlier in the release of membrane bound proteins into the aqueous layer. Chaotropic agent such as guanidine hydrochloride is commonly used to solubilize denatured insoluble proteins.
The extent of solubilization depends not only on the concentration of the protein present in the inclusion bodies but also on the concentration of the denaturant used. It has also been observed that the concentration of chaotropic agent required increases with increasing hydrophobicity of the protein. Usually the use of 6M GuHCl or 8M urea is sufficient to solubilize proteins present in the inclusion bodies. Urea, although less effective than guanidine in its ability to extract proteins in denatured state, is preferred as it is much cheaper. But the major draw back is that it forms cyanate ions at temperatures above 15°C that readily modifies free amino groups in proteins. This reaction can be mitigated by lowering the temperature to below 15°C and/or include tris-HCl buffer in the extraction medium.
In general, 6M-guanidine hydrochloride is found to be suitable for solubilization of most recombinant proteins present in E.coli inclusion bodies. Usually, inclusion bodies are extracted with 3-5 volumes of 6M-guanidine hydrochloride. When inclusion bodies are extracted with 6M GuHCl the resulting solution appears rather viscous making it difficult for direct column loading operations for further purification of the protein. To mitigate this problem, the inclusion
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bodies are pre treated with DNAase. However, such treatments could add to the cost of the process.
Simple dilution of the 6M GuHCl extract two fold with water results in some precipitate formation with reduction in viscosity. This reduction in viscosity is also accompanied by some loss of recombinant protein.
Summary of the invention:
To obviate the above drawbacks the instant invention provides for a novel multi-component (single-phase) system that gives complete extraction of the recombinant proteins from inclusion bodies. These extracts are less viscous and easy to apply to any chromatographic column. The efficacy and wider applicability of using this novel extractant with three different E.coli expressed recombinant proteins that form inclusion bodies have been demonstrated here.
Accordingly the instant invention provides for a process of extracting recombinant protein from inclusion bodies obtained from the E.coli comprising the step of:
solubilizing recombinant proteins expressed in the said E.coli with a buffer having a pH in the range of 7 to 11, wherein the said buffer contains guanidine hydrochloride, organic solvent and water, extracting the protein in temperatures ranging from 15°C to 40°C
The inclusion bodies contain recombinant proteins such as insulin precursor, interferon or HSP70.
The concentration of guanidine hydrochloride is 1.5 to 6 M preferably 4 to 6 M.
The said organic solvent in the buffer can be acetonitrile (20 to 50%(v/v)) or alcohol (20 to 50% (v/v))
The said alcohol can be methanol, ethanol, and a branched chain alcohol like isopropyl alcohol or a higher homologue.
The said organic solvent is a mixture of acetonitrile and alcohol in the ratio ranging from 100% of the former to 100% of the latter.
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The pH of the said buffer is in the range of 8 to 11.
The extraction is carried out at temperature in the range of 25°C to 40°C
The volume of extractant can be 3 to 10 fold over the inclusion body weight.
Composition of Guanidine hydrochloride-Acetonitrile-Water (GAWI) mixture:
The extractant consists of guanidine hydrochloride, acetonitrile and water (GAWI). The solid denaturant (guanidine hydrochloride) is highly soluble in water. The liquids namely water and acetonitrile are also freely miscible. But a mixture formed at certain ratios of these three components will exclude additional acetonitrile forming two-phase systems. The composition of the GAW will vary depending on the denaturant composition. For example to prepare 100ml of GAW mixture containing 6M denaturant 57.6g of guanidine hydrochloride is added to 30 ml of water and 30 ml of acetonitrile. The addition of water alters the composition of the system. Thus the ratio of water to acetonitrile is 1.0 at 6M-guanidine hydrochloride. This then defines the maximum organic solvent that one can use at this particular concentration of the denaturant. The GAW solution can be buffered with Tris-Cl to give a pH value of 8.0-11. Like wise, at lower denaturant concentrations also the novel 3 components will form a phase that would exclude added acetonitrile. The volume of water & organic solvent for some GuHCl concentrations are given in Table-1. GAW solutions varying in acetonitrile concentration other than the ones mentioned in Table-1 having guanidine hydrochloride concentrations in the range of 1-6M can also be prepared and used for extraction.
Composition of Guanidine hydrochloride- Alcohol-Water mixture (GAWII):
Similar to the 6M GAWI, 6M GAW II can be prepared with alcohols like methanol, ethanol, propanol, isopropanol or butanol. GAWII solutions with as much as 30% (v/v) alcohol have been prepared. But in the case of n-butanol, a maximum of 15-17% v/v can be used in the presence of 6M-guanidine hydrochloride. Further addition of n-butanol results in the exclusion of alcohol forming a 2-phase system. With other lower alcohols including isopropyl alcohol addition brings about increase in volume and dilution of the denaturant concentration.
Method of preparation:
The quantities of guanidine hydrochloride, acetonitrile & water required for making different GAW solutions are given in Table-1.
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Table-I GAW compositions for different guanidine concentrations

s.No. Weight of G Volume of Water Volume of acetonitrile Final volume Final cone of G in GAW
1. 57.6 g 30.0 mL 30.0 mL 100 mL 6.0 M
2. 36.0 g 33.8 mL 37.5 mL 100 mL 3.75 M
3. 28.3 g 36.5 mL 41.2 mL 100 mL 2.94 M
4. 20.9 g 38.0 mL 45.7 mL 100 mL 2.17 M
5. 14.4 g 38.0 mL 50.0 mL 100 mL 1.50 M
The instant invention will now be explained with examples:
Example 1:
Extraction of Recombinant human insulin precursor expressed in E.coli.
The gene for insulin precursor was assembled from chemically synthesized oligonucleotides. These oligos were annealed & ligated to give the proinsulin gene corresponding to B-chain-Lys-Arg-A-chain. This gene after appropriate restriction digestion was inserted into a pET vector. This plasmid harboring human insulin precursor gene was used to transform E.coli host. The expressed precursor is 8.3kD in size and composed of N-terminal His-tag in fusion with proinsulin peptide of the composition B-chain-Lys-Arg -A-chain.
E.coli clone harboring a gene for the human insulin precursor mentioned in its plasmid was allowed to grow at 37°C in a well-defined medium and induced with isopropyl thiogalactoside (IPTG), the inducer, for the expression of the human insulin precursor protein. The cells were harvested 3hrs after induction and were lysed in a Dyna Mill. The lysed cells were centrifuged at 10,000x g. The recombinant precursor protein expressed was found in pelleted inclusion bodies. This fraction was first washed with 50mM Tris-HCl buffer pH 8.0 containing 1% Triton X-100, 0.5M NaCl and 1M GuHCl. After washing, the inclusion bodies were extracted with 5 volumes of 6M GAWI-mixture (50mM Glycine-NaOH, pH 10.5) and centrifuged at l0000xg for 20 min.
The supernant is brownish in color and the pH of the extract is around 9.8. Both the pellet & the supernant were analyzed for the presence of the precursor protein by SDS-Polyacrylamide gel electrophoresis. The supernatant was found to contain the 8.3 kD recombinant insulin precursor protein, while the pellet was
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devoid of the precursor. Thus there is complete extraction of the desired recombinant protein from the inclusion bodies. The pH of the supernant was adjusted to 8.0 and used to further purify the 8.3kD protein by Ni-NTA affinity chromatography. The affinity purification of the insulin precursor was done after sulphonation of the protein or without this chemical modification. The chemically modified (sulphonated) or unmodified insulin precursor protein was bound to the column. The bound protein was eluted from the column using 50 mM acetate buffer pH 5.0 containing 3-6M guanidine hydrochloride. This showed that the presence of organic solvent does not interfere with the binding of the recombinant protein to the metal-chelate affinity matrix. The SDS- PAGE analysis showed the presence a major protein corresponding to the expected 8.3kD insulin precursor protein.
The initial extraction of the inclusion bodies can be performed with 6M GuHCl in buffer and after centrifugation, the supernant can be treated with organic solvent For example, 60 ml of acetonitrile is added to 100ml of 6M GuHCl extract. The resulting precipitate is removed by centrifugation. The clear supernant is a GAW solution containing 3.75M GuHCl.
The acetonitrile present in GAWI can be replaced by isopropyl alcohol to generate a 6M GAWII (Guanidine hydrochloride-alcohol-water mixture). This three-component system (single phase) also effectively extracted the recombinant insulin precursor protein from the inclusion bodies as judged by the protein profiles of the extracts in the SDS-polyacrylamide gel electrophorogram. In fact, this extract was clearer than the GAWI.
Instead of using the GAW solution for extraction, the inclusion bodies can also be first extracted with 6M GuHCl in buffer. The extract is then centrifuged to remove the insoluble material. To the supernant organic solvent is added in sufficient volume to make it a GAW solution. The precipitate formed is then removed by centrifugation at l000xg at 20°C The clear extract is used for further analysis & processing.
Example II -
Extraction of Recombinant interferon alpha 2b expressed in E.coli:
The gene for interferon alpha was assembled using chemically synthesized oligos and cloned into a pET vector. The E.coli cells were transformed with the vector and the clones obtained were analyzed for expression of the recombinant
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interferon. E.coli cells harboring the gene for interferon alpha 2b in their plasmid was allowed to grow at 37° C in a well-defined medium was induced with IPTG for the expression of the recombinant interferon. The cells were harvested and lysed in a Dyna Mill. The lysed cells were centrifuged at l0000x g. The recombinant interferon was found in the inclusion bodies. This fraction was washed with 50 mM Tris-HCl buffer pH 8.0 containing 1% Triton X-100, 0.5M NaCl and 1M GuHCl. After washing the inclusion bodies were extracted with 3-5 volumes of 6M GAWI or GAWII and centrifuged at l000x g for 20 min. An aliquot of the supernant was treated with trichloroacetic acid (final concentrationl0%v/v) to precipitate the proteins. The precipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis and found to contain 19.2kD interferon alpha 2b.
Example III -
Extraction of Yeast Heat Shock Protein 70 expressed in E.coli:
The SSA2 (HSP70) gene from S.cerevisiae was PCR amplified and cloned into pET20b vector. This plasmid was used to transform E.coli & the recombinant clones were analyzed for HSP70 expression. The expressed recombinant product was found to be associated with inclusion bodies. This 70 kD protein without any affinity-tag was also extracted with GAWI or GAWII effectively from the inclusion bodies as judged by the electrophoretic analysis of the GAW extract on SDS-polyacrylamide gel.
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We claim:
1. A process of extracting recombinant protein from inclusion bodies
extracted from the E.coli comprising the step of :
solubilizing recombinant proteins expressed in the said E.coli with a buffer having a pH in the range of 7 to 11, wherein the said buffer contains guanidine hydrochloride, organic solvent and water, extracting the protein at temperatures ranging from 15°C to 40°C
2. A process as claimed in claim 1 wherein the said inclusion bodies contain protein bodies such as insulin precursor, interferon and HSP70
3. A process as claimed in claim 1 wherein the concentration of said guanidine hydrochloride is 1.5 to 6 M
4. A process as claimed in claim 1 wherein the concentration of said guanidine hydrochloride is 4 to 6 M
5. A process as claimed in claim 1 wherein the said organic solvent in the buffer can be acetonitrile or alcohol.
6. A process as claimed in claim 1 wherein the said alcohol can be methanol, ethanol or a higher homologue.
7. A process as claimed in claim 1 wherein the said alcohol can be a branched chain alcohol (isopropyl alcohol) or a higher homologue.
8. A process as claimed in claim 1 wherein the said organic solvent is a mixture of acetonitrile and alcohol that range from 100% acetonitrile to 100% alcohol.
9. A process as claimed in claim 1 wherein the pH of the said buffer is in the range of 8 to 11.
10. A process as claimed in claim 1 wherein the extraction is carried out at temperature in the range of 25°C to 40°C.
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11. A process as claimed in claim 1 wherein the volume of extractant can be 3 to 10 fold over the inclusion body weight.
12. A process of extracting recombinant protein from inclusion bodies obtained from the E.coli substantially as herein described with reference to accompanying examples.
Dated this 22nd day of March 2005

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ABSTRACT
A process of extracting recombinant protein from inclusion bodies extracted
from the E.coli comprising the step of :
solubilizing recombinant proteins expressed in the said E.coli with a buffer having a pH in the range of 7 to 11, wherein the said buffer contains guanidine hydrochloride, organic solvent and water,
extracting the protein at temperatures ranging from 15°C to 40°C
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