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Exploring Vaccinia Virus as a Tool for Large‐Scale Recombinant Protein Expression
Author(s) -
Bleckwenn Nicole A.,
Bentley William E.,
Shiloach Joseph
Publication year - 2003
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0255762
Subject(s) - multiplicity of infection , microcarrier , vaccinia , recombinant dna , hela , virus , biology , green fluorescent protein , virology , cell culture , protein expression , microbiology and biotechnology , gene , genetics , biochemistry
Abstract A recombinant vaccinia virus was engineered to express enhanced green fluorescent protein (EGFP) under control of the T7 promoter using the VOTE expression system in HeLa cells. Infection of HeLa cells with this virus and induction with IPTG demonstrated the utility of this construct for easily measuring protein expression. This construct was used to evaluate several production parameters, specifically, multiplicity of infection (MOI), volume during infection, and serum concentration during the infection phase. In static culture, increasing multiplicity of infection was found to increase expression of EGFP up to a plateau around MOI of 1.0. Expression was also shown to increase with decreasing volume during the infection phase. Serum concentration during the infection phase was only marginally significant from 0 to 7.5%. Cytodex 3 microcarriers were found to have the best characteristics for HeLa cell growth. These cells were grown and infected in microcarrier spinner flask culture, and the maximum expression was 2.2 μg EGFP/(million cells at the time of infection), demonstrating the ability of this system to successfully express recombinant proteins at larger scale.