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Tuning cell cycle of insect cells for enhanced protein production

Paper ID Volume ID Publish Year Pages File Format Full-Text
23295 43430 2013 7 PDF Available
Title
Tuning cell cycle of insect cells for enhanced protein production
Abstract

•We identified the complete cyclin E (cycE) cDNA sequence from industrially relevant Trichoplusia ni (T. ni) derived High Five™ genomes, cloned the gene, and expressed and purified the protein.•We overexpressed cyclin E and found enhanced growth rate owing to additional cyclin E.•We employed RNAi to suppress cycE gene expression and found diminished cell growth.•We employed RNAi to “tune” cell cycle by arresting cells in G1 and doubled the yield of recombinant protein, as indicated by GFP expression.•The effect of RNAi on protein yield was found to be dose-dependent.

The eukaryotic cell cycle consists of many checkpoints during which certain conditions must be met before passing to subsequent stages. These safeguards ensure cells’ integrity and survival, but may also limit growth and protein synthesis in protein production processes. In this work, we employ metabolic engineering principles to “tune” the cell cycle to overcome checkpoint processes in order to facilitate faster cell growth, and independently, arrest the cell cycle in gap1 (G1) phase for greater protein productivity. Specifically, we identified the complete cyclin E (cycE) cDNA sequence from industrially relevant, Trichoplusia ni (T. ni) derived High Five™ genomes. We then both knocked down (through RNA interference; RNAi) and overexpressed (on an expression plasmid) cycE gene expression to tune the cell phenotype. We successfully up- and down-regulated cycE transcription, enhancing and hindering cell growth, respectively. We also measured the effects of titrated cycE expression on the cell cycle phase distribution. Finally, we investigated the dose-dependent effects of dsCycE on recombinant protein production using the baculovirus expression system and demonstrated a nearly 2-fold increase in expression of model protein (GFPuv).

Keywords
cycE, cyclin E; RNAi, ribonucleic acid interference; GFP, green fluorescent protein; CAT, chloramphenicol acetyl transferase; CHO, Chinese hamster ovary; ds, double stranded; G1, gap-1; G2, gap-2; M, mitosis; MOI, multiplicity of infection; S, DNA synthes
First Page Preview
Tuning cell cycle of insect cells for enhanced protein production
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 168, Issue 1, 10 October 2013, Pages 55–61
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering