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US 7,119,166 B2Granted
Universal procedure for refolding recombinant proteins
AssigneeOklahoma Medical Research Foundation
InventorXinli Lin
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AssigneeOklahoma Medical Research Foundation
InventorXinli Lin
Estimating the term…
A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are shown in Table I. The method has two key steps to unfold and then refold the proteins expressed in the inclusion bodies. The first step is to raise the pH of the protein solution in the presence of denaturing agents to pH greater than 9, preferably 10. The protein solution may be maintained at the elevated pH for a period of up to about 24 hours, or the pH immediately decreased slowly, in increments of about 0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or both steps used. In the preferred embodiment, purified inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione (GSSG), pH 10. The absorbance at 280 nm (OD280) of the protein solution is 5.0. This solution is rapidly diluted into 20 volumes of 20 mM Tris base. The resulting solution is adjusted to pH 9.0 with 1 M HCl and is kept at 4° C. for 24 hr. The pH is adjusted to pH 8.8 and the solution is kept at 4° C. for another 24 hrs. This process is repeated until the pH is adjusted to 8.0. After 24 hr at pH 8.0, the refolded proteins can be concentrated by ultrafiltration and applied to a gel filtration column for purification.
A method for refolding of a recombinant protein comprising
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From mammals: aspartic endopeptidases
Without change of the primary structure: by redox-reactions involving cystein/cystin side chains
Without change of the primary structure: by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating…
Tetrapeptides: with the first amino acid being acidic
Term ended 1 Aug 2021 (estimated)
The present invention is generally in the field of methods for manufacture of recombinant proteins, and especially in the field of refolding of recombinant proteins expressed in the inclusion bodies of procaryotic expression systems such as E. coli.
Expression of recombinant proteins with natural biological activity and structure, referred to as “proteomics”, becomes increasingly important with the completion of genomic sequencing for several organisms and the near completion of human genome sequencing. One aspect of proteomics is to express large amounts of protein for structural and functional studies, as well as for commercial applications. The least expensive and most efficient way to express recombinant proteins is to express the proteins in E. coli. Proteins are expressed either intracellularly or secreted into the periplasmic spaces. In the former case, the proteins are often deposited in inclusion bodies, especially if the protein has disulfide bonds.
However, one of the problems in expressing mammalian proteins in E. coli is that most of the expressed proteins form insoluble inclusion bodies. While this problem can be circumvented by using various mammalian or insect expression systems, growing E. coli is faster and less expensive compared to mammalian and insect cultures. Moreover, some proteins are toxic to the host when expressed in their native forms, thus expression as insoluble inclusion bodies is the only way to obtain large quantities of recombinant proteins. Importantly, high levels of expression can be achieved for most proteins. 400 to 600 mg of inclusion bodies per liter of bacterial culture can routinely be achieved, with up to 9,700 mg/L having been reported using this method (Jeong K L; Lee S Y, 1999. Appl. Environ. Microbiol 65:3027–32). Inclusion bodies can be easily purified to greater than 90% with a simple freeze/thaw and detergent washing procedure.
A. Reagents:
ZB media: 10 g N-Z-Amine A, 5 g NaCl/L
LB media: 10 g Tryptone, 5 g Yeast extract, 10 g NaCl/L, pH 7.5