Expression refolding

LJ Frank, D Wisniewski, GG Hammond, J Hermes, A Marcy, PM Cameron. High-yield expression, refolding, and purification of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus strain 27R. Prot Expr Purif 6 671-678, 1995. 

In conclusion, the supply of pure, natively folded chemokine proteins is vital for a variety of basic, translation and cHnical research programs. Our structure-function studies and drug development efforts have motivated the development of the robust protocol for recombinant chemokine expression, refolding, purification, and vahdation presented here. Finally, many additional factors must be considered when manufacturing a product under cGMP guidehnes that may not be obvious to personnel in research settings. 

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

Lowe, P. A. and Rhind, S. K., Solubilization, refolding, and purification of eukaryotic proteins expressed in E. coli, in Protein Purification — Micro to Macro, Burgess, R., Ed., Alan R. Liss, New York, 1988, 429. 

P. fluorescens has well-developed mechanisms for the secretion of proteins into the periplasm which facilitates S—S bond formation and proper N-terminal processing. It also allows one to reduce the formation of inclusion bodies and, thus, the additional costs caused by refolding processes. Proteolytic degradation of the expressed protein is also low, and very high... 

Tsouloufis et al.8 used an ELISA to assess the refolding of a recombinant subunit of the extracellular domain of the human muscle acetylcholine receptor expressed in E. coli. The plates were coated with refolded or unfolded protein and then reacted with conformationally dependent MAbs. The use of specific... 

Fusion protein pull-down assays involve the overexpression of bait and/or fusion proteins in bacteria. Often, the expressed fusion proteins are localized in occlusion bodies and not readily soluble under nondenaturing conditions. The expressed proteins can be extracted using urea, sonication, sodium dodecyl sulfate (SDS), or a combination of all the three. The net result is the denaturation of the recombinant protein and it may need to be refolded if the interaction domain is conformationally dependent. A major advantage of the pull-down assay is that high concentrations of proteins can be easily generated thus favoring protein association for a reversible equilibrium between two proteins. 

For Ras proteins, there is no chaperone protein available or known to solubilize the lipidated protein thus the refolding of, for example, lipidated N- and H-Ras is not feasible. However, lipidated K-Ras 110 can be generated via expressed protein ligation (EPL), since the polybasic C-terminal region of this protein helps to solubilize the protein even when farnesylated and facilitates the purification (Scheme 36). ... 

Heat-shock proteins (Hsps) are proteins expressed virtually in all organisms as a response of exposure to a stress, such as elevated temperature (fever), protein degradation, mechanical or chemical stress. As chaperone proteins they are concerned with the intracellular folding and refolding, assembly and translocation of damaged proteins. 

The successful expression of recombinant plant peroxidases such as HRP C has been a major focus of research in a number of laboratories. Three synthetic HRP C genes based on the amino acid sequence determined by Welinder (36, 47) were synthesized independently in order to initiate this work (63-65). A number of different expression systems have been evaluated (64, 66-73), a summary of which is presented in Table I. Refolding of recombinant HRP C isolated from inclusion... 

Fortuitous ligands. Ligands from the expression host (e.g. fatty acids from Escherichia coll) could be noncovalently bound, e.g. in the active site of the overexpressed target protein. This is a serious, but rare, problem and may require an additional refolding step in the protein purification protocol. The presence of any noncovalently bound ligands in the target protein can be checked by nondenaturing ESI-MS. 

G Zhao, TI Meier, J Hoskins, SR Jaskunas. Penicillin-binding protein 2a of Streptococcus pneumoniae, expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme. Prot Expr Purif 16 331-339, 1999. 

QM Wang, RB Johnson, GA Cox, EC Villarreal, LM Churgay, JE Hale. Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli. J Virol 72 1683-1687, 1998. 

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