Transfected insect cells in suspension culture rapidly yield moderate quantities of recombinant proteins in protein-free culture medium

Protein Expression and Purification
Patrick Farrell, Kostas Iatrou

Abstract

Methodology to rapidly express milligram quantities of recombinant proteins through the Lipofectin-mediated transfection of insect cells in small-scale, protein-free suspension culture is presented. The transfection phase in suspension culture was first optimized using the green fluorescence protein coupled with FACs analysis to examine the effect of variables such as the transfection media, duration, and cell density on transfection efficiency and expression level. The recombinant protein production phase was optimized using secreted alkaline phosphatase (SEAP) as a reporter protein to evaluate the cell seeding density and harvest time. Using this method, 5 secreted, 2 intracellular, and 1 chimeric protein were expressed at levels ranging from 6 to 50 mg/L. Furthermore, the ability to purify over 2 mg of His(6)-tagged SEAP by immobilized metal affinity chromatography from 50 mL insect cell culture medium to greater than 95% purity was also demonstrated. This method is suitable for scale-up and high-throughput applications.

References

Apr 21, 1999·Current Opinion in Biotechnology·F Wurm, A Bernard
Dec 10, 1999·Biotechnology Progress·M B KeithL A Behie

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Citations

Apr 16, 2017·Journal of Bioscience and Bioengineering·Keita MoriHideki Yamaji
Nov 26, 2008·Protein Expression and Purification·Sabine Geisse
Feb 29, 2008·Biotechnology and Bioengineering·Marina SkarjinskaiaVidadi Yusibov
Jul 14, 2007·Insect Biochemistry and Molecular Biology·Zhiyan LiuBryony C Bonning

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