Interleukin 2, refolding

Brent Pollock (Biomira Inc.) presented a talk on the rational development of a second-generation production process for recombinant human interleukin-2 that incorporates recent advances in cell expression systems, fermentation optimization, protein extraction, refolding, and purification. A key concept of this talk was to know thy protein. The driving forces for process changes were regulatory concerns/robustness, economics, the ability to use standard equipment, and the elimination of bottlenecks in the process. 

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). 

Prestrelski et al. (66) used IR to optimize lyophilization conditions for interleukin-2 (IL-2). The authors were able to show that proteins can unfold during lyophilization, and that altering the lyophilization conditions determines whether the proteins refold into the original conformation upon reconstitution. Dong et al. (67) provide a comprehensive review of the use of infrared spectroscopy for the study of the effects of lyophilization on proteins. 

Proteins that contain disulfide bonds often fold slowly in vitro because the oxidation and coixect pairing of the cysteine residues becomes the rate-limiting step and the bonds formed ai e not always the coiiect ones. Many proteins, especially those that are secreted by eu-kaiyotes, aie stabilized by disulfide bonds. Examples of such proteins include those used for medical or biotechnological puiposes, such as interleukins, IFNs, antibodies and then fragments, insulin, TGF, and many toxins and proteases. Expression of recombinant proteins as inclusion bodies in bacteria can be a very efficient way to produce cloned proteins, as long as the inclusion body protein can be successfully refolded. 

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