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Refolding of Recombinant Proteins

a recombinant protein and refolding technology, applied in the field of refolding of recombinant proteins, can solve the problems of cellular machinery, cellular machinery, and often expensive media for animal cell culture,

Inactive Publication Date: 2008-05-29
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides a method for recovering and purifying refolded recombinant proteins from prokaryotic host cells, particularly bacteria. The method involves solubilizing and refolding the protein in a buffered solution, using a combination of urea, arginine, cysteine, and reducing agents. The refolded protein can then be recovered using a variety of techniques such as clarification and chromatographic supports. The invention also provides methods for refolding and purifying recombinant proteins from manufacturing or industrial scale cell cultures.

Problems solved by technology

Additionally, animal cell cultures often require expensive media containing growth components that may interfere with the recovery of the desired protein.
However, bacterial expression systems such as E. coli lack the cellular machinery to facilitate proper refolding of the proteins and generally do not result in the secretion of large proteins into the culture media.
Additionally, refolding often produces misfolded and disulfide-linked dimers, trimers, and multimers.
These methods seek to eliminate random disulfide bonding prior to coaxing the recombinant protein into its biologically active conformation through its other stabilizing forces and may not eliminate improperly folded intermediates, provide homogenous populations of properly folded product, or provide sufficient amounts of the properly folded product.
However, these methods are not always efficient or sufficient to produce quantities of protein product.

Method used

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  • Refolding of Recombinant Proteins
  • Refolding of Recombinant Proteins
  • Refolding of Recombinant Proteins

Examples

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Effect test

example 1

Solubilizing and Refolding of Recombinant human VEGF Expressed in Escherichia coli

Methods

[0116]Plasmid for VEGF165 expression—The plasmid pVEGF171 is designed for the expression of human VEGF165 (see, e.g., Leung et al., (1989) Science, 246: 1306-1309) in the E. coli periplasm. Transcription of the VEGF coding sequence is placed under tight control of the alkaline phosphatase (AP) promoter (see, e.g., Kikuchi et al., (1981) Nucleic Acids Research, 9:5671-8), while sequences required for translation initiation are provided by the trp Shine-Dalgarno region (see, e.g., Yanofsky et al., (1981) Nucleic Acids Research, 9:6647-68). The VEGF coding sequence is fused downstream of the bacterial heat-stable enterotoxin II (STII) signal sequence (see, e.g., Lee et al., (1983) Infect. Immun. 42:264-8; and, Picken et al., (1983) Infect. Immun. 42:269-75) for subsequent secretion into the E. coli periplasm. Codon modifications in the STII signal sequence provide for an adjusted translation level...

example ii

ingle Step Solubilization and Refolding of Recombinant human VEGF Expressed in Escherichia Coli

[0120]Solubilization and Refolding—The pellet is suspended in 10-39 liter volumes of refolding buffer (in this case termed “combo buffered solution”) for every kg of cell pellet, where the combo buffered solution contains 1 M Urea, 15 mM cysteine, 0.5 or 2 mM DTT, 100 mM arginine, 10 mM Tris or CHES, 5 mM EDTA, pH 9.5-10.5, final concentration. See FIG. 6 for the effect of urea and arginine addition in the refolding buffered solution. FIG. 6 shows the results of a 1-step pellet refold (combo buffered solution) as described in this example at pH 9.5 for 15 hours at room temperature. The denaturant concentrations are varied as follows: (1) 1 M urea and 100 mM arginine; (2) 1 M urea (and 0 mM arginine); (3) 2 M urea (and 0 mM arginine), while all the other buffer components (e.g., Tris or CHES, DTT, etc.) remain in the same concentration. The VEGF titer extracted from these is equivalent as ...

example iii

Non-Pellet Refolding of Recombinant Protein

[0122]Escherichia coli whole cell broth producing recombinant protein is homogenized in a model 15 M laboratory homogenizer Gaulin 15M (small scale) or M3 (large scale) (Gaulin Corporation, Everett, Mass.) and diluted 1:4 (v / v) in refolding buffer per volume of homogenate and sparging with air at a mass transfer coefficient kLa=0.004 to 0.01 min−1, (e.g., For 2.5L vessel with marine type impeller, the air sparging rate would be 0.3 to 3 cc / min / L, or 0.3-1 cc / min / L, or 1 cc / min / L, or 3 cc / min / L while mixing is 200-400 rpm). The refolding buffer contains 1 M Urea, 15 mM cysteine, 2 mM DTT, 100 mM arginine, 10 mM Tris or CHES, 5 mM EDTA, pH 9-10, final concentration. Refold incubation is conducted at room temperature for 3-24 hours. Optionally, VEGF can be stabilized in the refold buffer by adding nitrogen in place of air after 3 hours refold incubation. The folding is monitored by cation exchange HPLC, rpHPLC chromatography, and / or Heparin HP...

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Abstract

Processes are provided for recovering and purifying refolded recombinant proteins produced in heterologous host cells, which includes the step of refolding the protein in a high pH buffer.

Description

RELATED APPLICATION[0001]This application claims priority to and the benefit of U.S. Provisional Application Ser. No. 60 / 830,831, filed Jul. 14, 2006 specification of which is incorporated herein in its entirety.FIELD OF THE INVENTION[0002]This invention relates to methods for obtaining heterologous recombinant proteins produced in cell culture. The invention includes methods for recovering and purifying refolded recombinant proteins that have been produced in prokaryotic host cells and are present in these cells, typically in the periplasmic or intracellular space. The recombinant proteins produced in prokaryotic host cells can also be found as soluble proteins or as a mixture of soluble and insoluble proteins.BACKGROUND[0003]Many therapeutically relevant recombinant proteins are produced in a variety of host organisms. Most proteins can be expressed in their native form in eukaryotic hosts such as CHO cells. Animal cell culture generally requires prolonged growing times to achieve...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K1/00C07K1/14C07K1/36C07K14/475
CPCC07K1/36C07K1/20C07K1/1136C07K14/00C07K1/1133C07K1/14C07K1/16C07K14/475
Inventor PIZARRO, SHELLYSANCHEZ, AILENSCHMELZER, CHARLES H.
Owner GENENTECH INC
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