title: Tuning cell cycle of insect cells for enhanced protein production.
abstract: 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).
authors: Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA; Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD 20742, USA.
authors: Hsuan-Chen
authors: HC
authors: Wu
authors: Colin G
authors: CG
authors: Hebert
authors: Chi-Wei
authors: CW
authors: Hung
authors: David N
authors: DN
authors: Quan
authors: Karen K
authors: KK
authors: Carter
authors: William E
authors: WE
authors: Bentley
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D007313:Insecta; D011506:Proteins / Q000378:metabolism*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D011506:Proteins
keywords: Baculovirus; CAT; CHO; Cell cycle; Chinese hamster ovary; DNA synthesis; G1; G2; GFP; M; MOI; RNA interference; RNAi; RT-PCR; Recombinant protein; S; T. ni; Trichoplusia ni; chloramphenicol acetyl transferase; cycE; cyclin E; double stranded; ds; gap-1; gap-2; green fluorescent protein; mitosis; multiplicity of infection; reverse transcription polymerase chain reaction; ribonucleic acid interference; single stranded; ss