Protein renaturation

Two features of recombinant production in particular can impact very significantly upon the approach subsequently taken to purify the recombinant product inclusion body formation and the incorporation of purification tags. The processes of inclusion body formation, recovery and recombinant protein renaturation have been considered in Chapter 5. Once the recombinant protein has been refolded, additional purification (if required) follows traditional lines. 

This type of kinetic situation sometimes occurs in protein renaturation experiments, in which the kinetics are often monitored by fluorescence changes. The biphasic traces cannot be resolved in such circumstances unless the quantum yield of the transient intermediate is known, so that its absolute concentration can be determined. 

Some bioproducts are derived from fermentation by living organisms but immobilized enzymes can also catalyze a biotransformation. If the product is extracellular, then the initial steps of recovery include removal of the cells and other particulate matters from the broth. If the product is intracellular, it would be necessary to lyse the cells to release the product into the broth. The cell debris is then separated before the product is recovered from the broth. In certain cases, proteins produced as IBs need to be solubilized and the proteins renatured before further recovery steps. In the case of biotransformed products, the immobilized cells or enzymes and their support need to be removed initially. 

This book attempts to bridge the gap between fundamental and applied studies on protein folding. Some of the issues addressed include in vivo protein folding, protein aggregation and inclusion body formation, elucidation of the folding pathway, characterization of folding intermediates, and practical considerations in protein renaturation. 

The peak fractions (usually 3-8), determined by protein assay, are diluted fivefold with buffer A. In GM-CSF-PE38KDEL the yield at this point was 12.3 mg from 37 mg of total protein renatured in step 2, Subheading 3.5. (13). 

The fractions from the sizing column are analyzed by reducing SDS-PAGE, pooled, and stored at -80°C. A typical yield is 5-10% of total recombinant protein renatured in step 2, Subheading 3.5. 

Molecular chaperones, traditionally proteins, facilitate the folding of proteins by interacting non-covalently with non-native folding intermediates and not with either the native or totally unfolded protein. When folding is complete, molecular chaperones are not required to maintain proper conformation. However, molecular chaperone function is not restricted to proteins (Bogdanov et ah, 1996 Ellis, 1997). Specific lipids are able to interact with partially folded proteins in a transient manner in either de novo protein folding or protein renaturation in vitro similar to that of protein molecular chaperones. 

EAN has been used as a solvent in a number of protein renaturation and crystallization studies. " " The hydro-phobic, ionic nature and ability to form hydrogen bonds makes EAN a good solvent for proteins. The PILs used in the biological field have nearly exclusively involved EAN, with the exception of the antimicrobial work by Pemak et al. using imidazolium PILs. ... 

Summers CA, flowers RA (2000) Protein renaturation by the liquid organic salt ethylammonium nitrate. Protein Sci 9 2001-2008... 

Besides the modification of the protein itself, variations of external parameters such as ionic strength, presence of anions or cations, and pH, can modify the rate and yield of protein renaturation. Ahmed and co-workers (1975) investigated the action of some effectors on the refolding of reduced RNase in the glutathione regeneration system. Ahmed and co-workers (1975)... 

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