Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment

Protein Engineering, Design & Selection : PEDS
P BelinA Ménez

Abstract

We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating th...Continue Reading

Citations

Jun 7, 2011·Protein Engineering, Design & Selection : PEDS·Meghan A LockardGeoffrey S Waldo
Jan 5, 2010·Biotechnology and Applied Biochemistry·Yanina R SevastsyanovichJeffrey A Cole
Jul 16, 2011·Analytical Biochemistry·Marla D SwainEllen R Goldman
Jan 3, 2007·Journal of Molecular Biology·Ellen WangPirkko Heikinheimo
Oct 11, 2011·Mechanisms of Development·Meytal HadadF Shira Neuman-Silberberg
Jul 30, 2018·Journal of Bioscience and Bioengineering·Panwad RitthisanHideo Nakano

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