Polyethylene glycol enhanced protein refolding.
Previous studies on the refolding of recombinant bovine carbonic anhydrase B (CAB) indicated that polyethylene glycol (PEG) significantly enhanced the recovery of active protein by reducing aggregation. To further test the ability of PEG to enhance refolding, three recombinant human proteins, deoxyribonuclease (rhDNAse), tissue plasminogen activator (rhtPA), and interferon-gamma (rhIFN-gamma) were refolded in the presence of PEG (3350 MW). rhDNAse produced from CHO cells was denatured in 7.2 M urea and refolded by rapid dilution to 4.0 M urea and 0.20 mg/ml protein. When a final PEG to rhDNAse molar ratio of 5 to 1 (0.1 milligram PEG, 3350 MW) was used in the dilution buffer, refolding was improved by 30% to yield complete recovery of active protein. Impure E. coli derived inclusion body preparations of rhDNAse were solubilized in 8 M urea and refolded by dilution to 4 M urea and 0.10 mg/ml protein. Refolding with a dilution buffer which yielded a final PEG to rhDNAse molar ratio of 10 to 1 (0.1 milligram PEG, 3350 MW) resulted in a three-fold increase in the recovery of active protein. When PEG was used in the dilution buffer, aggregation of rhDNAse did not occur during refolding in either case. rhtPA produced from CHO cells was denatured in 5 M guanidine hydrochloride (GuHCl) and refolded by rapid dilution to 0.10 M GuHCl and 0.20 mg/ml protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Genentech, Inc., S. San Francisco, CA 94080.
J L
JL
Cleland
S E
SE
Builder
J R
JR
Swartz
M
M
Winkler
J Y
JY
Chang
D I
DI
Wang
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry*; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D007371:Interferon-gamma; D003851:Deoxyribonucleases; D010959:Tissue Plasminogen Activator