Correctly folded protein

CAT folding reporter antibiotic resistance correctly folded proteins impart antibiotic resistance to organism via a chloramphenicol acetyl-transferease fusion tag Maxwell, 1999... 

Only correctly folded proteins can perform their biological function correctly. This complicated process is currently under active investigation a whole class of folding helper proteins, the chaperones [2], are used by Nature to prevent misfolding events with their often dramatic consequences (TSE, Alzheimer s disease, etc., vide infra). 

Proteins that contain multiple cysteines are difficult to refold after high-level production in E. coli because of the formation of incorrect disulfide bonds. Replacement of individual cysteines or specific disulfide bonding pairs without compromising the functional activity of the protein can result in increased yield of the correctly folded protein. This technique has been applied successfully to interleukin-2 (IL-2 Wang et al, 1984), human fibroblast interferon (Mark et al, 1984), and basic fibroblast growth factor (b-FGF Rinas et al, 1992). 

Low solubility or improper folding may sometimes hamper the use of enzymes, particularly when expressed in a non-native host. A method of expressing proteins with a C -terminal GFP fusion to use fluorescence as a measure of the amount of correctly folded protein has been introduced (44). DNA shuffling produced variants of ferritin that showed increased solubility, even when they were recloned without the GFP fusion. This assay has been used to produce proteins for X-ray crystallography structure determination (45). The protein nucleoside diphosphate kinase from Pyrobaculum aerophilum is insoluble when expressed in E. coli, but after DNA shuffling, a functional variant with six mutations was found to have 90% solubility, which enabled its crystallization, and its structure was determined. 

The amount of time needed to search from a random structure with more than 1,200 atoms to a correctly folded protein can be tmeconomical or impractical. For larger structures (>1,200 atoms), SA is often used to refine approximate... 

In theory, renaturation may be accomplished by removal of the denaturing agent. However, in practice, the problem is far more complex and suboptimal renaturation can often lead to protein aggregation and/or inactivation with a low recovery of correctly folded protein. 

Georgiou, G. and Valax, P. (1996). Expression of correctly folded proteins in Escherichia coli. Curr. Opin Biotechnol. 7, 190-197. 

C.B. Anfinsen, Principles that Govern the Folding of Protein Chains. Science, 181, 223-230, 1973. The Anfinsen principle is that the sequence of a protein dictates the full three-dimensional structure that it would form, that is, sequence dictates protein folding and assembly. The need for molecular chaperones suggests that the correctly folded protein, the lowest energy structure, is not always the result and that the problem arises out of improper hydrophobic associations. Interestingly, the... 

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