title: Efficient expression of secreted proteases via recombinant BacMam virus.
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.
authors.affiliation: GlaxoSmithKline, Discovery Research Biology, Department of Gene Expression and Protein Biochemistry, Gunnels Wood Road, Stevenage SG1 2NY, UK. martin.j.scott@gsk.com
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Scott
authors.forename: Sundip S
authors.initials: SS
authors.lastname: Modha
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Rhodes
authors.forename: Neil M
authors.initials: NM
authors.lastname: Broadway
authors.forename: Phil I
authors.initials: PI
authors.lastname: Hardwicke
authors.forename: Huizhen J
authors.initials: HJ
authors.lastname: Zhao
authors.forename: Karen M
authors.initials: KM
authors.lastname: Kennedy-Wilson
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Sweitzer
authors.forename: Steve L
authors.initials: SL
authors.lastname: Martin
mesh_terms: D016367:Baculoviridae / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D010447:Peptide Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D010447:Peptide Hydrolases