High recovery of prochymosin from inclusion bodies using controlled air oxidation.
Refolding of proteins from inclusion bodies is a field of increasing interest for obtaining large amounts of active enzymes. Consequently, the development of inexpensive and scalable processes is required. This is particularly challenging in the case of eukaryotic proteins containing cysteines, which may form disulfide bonds in the native active protein. Previous studies have shown that the formation of disulfide bonds is essential for the refolding of prochymosin. In this work we demonstrate that air oxidation can be efficiently used for the refolding of prochymosin and that 48% of the unfolded protein can be recovered as active enzyme at a final protein concentration of 0.8 mg/ml. Refolding of the protein strictly correlates with the change in pH of the refolding solution. We were able to follow the degree of oxidative renaturation of the prochymosin by simply measuring pH. Thus, the scaling up of the refolding system under controlled conditions was easily achieved. Analyses of different substances as folding aids indicate that the use of L-arginine or neutral surfactants improves the recovery of active protein up to 67% of the initial protein. The overall results indicate that prochymosin can be efficiently and inexpensively refolded with high yields by controlled air oxidation.
Microbiology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK), Argentina.
Hugo G
HG
Menzella
Hugo C
HC
Gramajo
Eduardo A
EA
Ceccarelli
D000388:Air; D012085:Chymosin / Q000302:isolation & purification* / Q000378:metabolism*; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D010100:Oxygen / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects
D004792:Enzyme Precursors; C032443:prorennin; D012085:Chymosin; D010100:Oxygen