Efficient expression of secreted proteases via recombinant BacMam virus

Protein Expr Purif. 2007 Mar;52(1):104-16. doi: 10.1016/j.pep.2006.09.017. Epub 2006 Oct 26.

Abstract

Baculovirus vectors engineered to contain mammalian cell-active promoter elements have been described as an efficient method for transduction of a broad spectrum of human cell lines at high frequency. In the first large-scale comparative study of secreted protein production using these viral vectors, we have evaluated production of 16 recombinant enzymes--specifically, we exploited these viral vectors, termed 'BacMam' viruses, to drive expression of a panel of proteases selected from all four major mechanistic classes, including secreted, lysosomal, endosomal, and type I transmembrane proteins. To allow a generic purification strategy, coding sequences were truncated to remove transmembrane and/or subcellular retention signals before introduction, in parallel, into a C-terminally Fc-tagged BacMam transfer vector. BacMam viruses were generated and subsequently evaluated for expression of Fc-tagged protein in virus-transduced HEK-F cells. The common Fc-tag enabled single-step affinity purification of secreted recombinant protein from the culture medium. Yields were excellent, with 14 of 16 genes expressed producing 10-30 mg or more purified protein per litre of culture using standardised transduction conditions. At this level, reagent demands for a typical protease high-throughput screen (HTS) could be met from expression cultures as small as 0.1-0.5 L. Our results indicate this expression system offers a highly efficient and scaleable method for production of enzymatically-active secreted proteases and may therefore represent a novel method of protein production for other secreted enzymes with significant advantages over the diverse approaches in current use.

MeSH terms

  • Baculoviridae / genetics*
  • Baculoviridae / growth & development
  • Cell Line
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Genetic Vectors
  • Humans
  • Kinetics
  • Peptide Hydrolases / genetics*
  • Peptide Hydrolases / isolation & purification
  • Peptide Hydrolases / metabolism
  • Plasmids
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism*
  • Transfection

Substances

  • Recombinant Proteins
  • Peptide Hydrolases