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Improving Renaturation of Proteins from Inclusion Bodies
Author(s) -
Kotlarski Nicholas,
O'Neill Brian K.,
Dunlop Eric H.,
Francis Geoffrey L.
Publication year - 2002
Publication title -
developments in chemical engineering and mineral processing
Language(s) - English
Resource type - Journals
eISSN - 1932-2143
pISSN - 0969-1855
DOI - 10.1002/apj.5500100610
Subject(s) - dilution , chemistry , yield (engineering) , reaction rate constant , protein aggregation , mixing (physics) , monomer , escherichia coli , chromatography , kinetics , growth rate , crystallography , biophysics , biochemistry , materials science , organic chemistry , biology , thermodynamics , physics , geometry , mathematics , quantum mechanics , gene , metallurgy , polymer
The effect of varying mixing rate and mode of dilution during refolding of proteins is poorly characterized. However, these two physical parameters are important design considerations for large‐scale processes. In this paper, an investigation of the refolding of an analogue of Insulin‐like Growth Factor I (LongR 3 IGF‐I; Wells et al., 1994) is presented. Partially purified inclusion bodies of the protein were isolated from Escherichia coli (E. coli), dissolved following a standardized protocol and refolded in either batch mode under different mixing rates or fed‐batch mode at a protein concentration of up to 2 g L −1 . Rate constants for a widely accepted kinetic model of refolding were regressed to transient protein concentration as a key measure of performance. It was found that mixing rate did not significantly alter a first‐order rate constant of correct refolding, k N = 0.17 ± 0.05 min −1 . However, it did increase an apparent second‐order rate constant of aggregation, K A , from approximately 1.6 (without stirring) to 3.3 L g −1 (rapidly stirred). Refolding at high protein concentration in fed‐batch mode gave approximately 5‐fold greater maximal yield of renatured protein than in batch mode. This increase in yield of active monomer was accompanied by a reduction in the amount of aggregate species resolved by non‐reducing SDS‐PAGE. Results indicate that gradual addition of denatured protein to give conditions that promote refolding is superior to rapid batch dilution, but an increased rate of mixing reduces the yield of active product by increasing the rate of aggregation. The kinetic model was shown to give a poor description of refolding at high protein concentration in both batch and fed‐batch modes.