Efficient expression of secreted proteases via recombinant BacMam virus
| Autor: | Andrew D. Rhodes, Phil Hardwicke, Sharon Sweitzer, Sundip Modha, Huizhen J. Zhao, Karen M. Kennedy-Wilson, Steve L. Martin, Martin J. Scott, Neil Broadway |
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| Rok vydání: | 2006 |
| Předmět: |
Proteases
Genetic Vectors Proteolytic enzymes BacMam Biology Transfection Molecular biology Transmembrane protein Recombinant Proteins law.invention Cell biology Viral vector Cell Line Transduction (genetics) Kinetics law Cell culture Recombinant DNA Escherichia coli Humans Baculoviridae Biotechnology Peptide Hydrolases Plasmids |
| Zdroj: | Protein expression and purification. 52(1) |
| ISSN: | 1046-5928 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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