InsectDirect System: rapid, high-level protein expression and purification from insect cells
| Autor: | Mark Mehler, Kathryn H. Loomis, Michael M. Batenjany, Shou C. Wong, Anthony Grabski, Keith W. Yaeger, Robert E. Novy |
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| Rok vydání: | 2004 |
| Předmět: |
Proteomics
Genetic Vectors Sf9 Biology Spodoptera Transfection Biochemistry Polymerase Chain Reaction Open Reading Frames Structural Biology Heat shock protein Genetics Protein biosynthesis Animals Humans Cloning Molecular Expression vector fungi Proteins General Medicine Molecular biology Fusion protein Open reading frame Target protein |
| Zdroj: | Journal of structural and functional genomics. 6(2-3) |
| ISSN: | 1345-711X |
| Popis: | A fundamental challenge in high-throughput (HT) expression screening is to rapidly identify the appropriate expression system for many targets in parallel. Known or unknown open reading frames (ORFs) are typically amplified by PCR and then cloned into a variety of vectors, producing recombinants used to direct target protein expression in Escherichia coli, insect cells, mammalian cells, or yeast. To facilitate rapid expression and purification in Spodoptera insect cells (Sf9), we developed transient expression vectors that include an enterokinase cleavage site immediately upstream of a ligation-independent cloning site (Ek/LIC). We also developed a high-efficiency insect cell transfection reagent, and automation-compatible fusion protein purification system for insect cells to facilitate expression screening and protein production. Positive clones identified from the small-scale screening were subjected to a larger scale production. Using this InsectDirectTM approach, we successfully expressed milligram quantities of different human proteins including heat shock proteins, phospholipases, and protein kinases. |
| Databáze: | OpenAIRE |
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