Maximizing recovery of native protein from aggregates by optimizing pressure treatment

Brian G. Lefebvre, Matthew J Gage, Anne Skaja Robinson

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 ± 0.9 to 47.4 ± 19.6 μg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.

Original languageEnglish (US)
Pages (from-to)623-629
Number of pages7
JournalBiotechnology Progress
Volume20
Issue number2
DOIs
StatePublished - Mar 2004
Externally publishedYes

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ASJC Scopus subject areas

  • Food Science
  • Biotechnology
  • Microbiology

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