Chaperone-mediated refolding of recombinant prochymosin.
It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.
Wei
C
C
Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Zhang
Y
Y
Yang
K
K
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002417:Cattle; D018833:Chaperonins; D012085:Chymosin / Q000737:chemistry*; D004792:Enzyme Precursors / Q000737:chemistry*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin; D018833:Chaperonins; D019704:Protein Disulfide-Isomerases