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Polyethylene Glycol Enhanced Protein Refolding

Bio/Technology volume 10, pages 1013–1019 (1992)Cite this article

Abstract

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–γ) 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 g/l 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 g/l 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. Dilution to these conditions with a buffer containing a final molar ratio of PEG to rhtPA of 20 to 1 (0.19 g/I PEG, 3350 MW) resulted in a two fold increase in the recovery of proteolytically active rhtPA and protein aggregation was again prevented. E. coli derived rhIFN–γ was denatured in 4 M GuHCl and refolded by rapid dilution to 0.5 or 1.0 M GuHCl and 1.0 mg/ml protein. A PEG to rhIFN–γ molar ratio of 2 to 1 (0.38 g/l PEG, 3350 MW) increased the yield of active protein from 5% to greater than 30%. A good correlation was observed between the protein hydrophobicity and the stoichiome–tric concentration of PEG required to achieve a maximal increase in recovery of active protein. These refolding studies reveal that PEG has significant potential for enhancing the recovery of active proteins produced in heterologous hosts.

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Author notes

  1. Daniel I. C. Wang: Corresponding author.

Authors and Affiliations

  1. Pharmaceutical Research & Development, Genentech, Inc., S. San Francisco, CA, 94080

    Jeffrey L. Cleland

  2. Recovery Process Research & Development, Genentech, Inc., S. San Francisco, CA, 94080

    Stuart E. Builder & Marjorie Winkler

  3. Fermentation Process Research & Development, Genentech, Inc., S. San Francisco, CA, 94080

    James R. Swartz & Judy Y. Chang

  4. Biotechnology Process Engineering Center, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139

    Daniel I. C. Wang

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  4. Marjorie Winkler
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  5. Judy Y. Chang
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Cleland, J., Builder, S., Swartz, J. et al. Polyethylene Glycol Enhanced Protein Refolding. Nat Biotechnol 10, 1013–1019 (1992). https://doi.org/10.1038/nbt0992-1013

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