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

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 the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

“…We envision that the immobilized bead assay described here will be applied to a broad scope of biological problems in addition to protein crystallization target selection and the identification of stably assembled complexes. One such application that has yet to be explored is host strain engineering, where the increased functional or soluble expression of target host proteins can be used to select for chromosomal mutations that improve protein production (Belin et al ., 2004; Massey-Gendel et al ., 2009). …”

Sense and nonsense from a systems biology approach to microbial recombinant protein production

“…However, it did provide the starting point for the isolation of an improved strain with an uncharacterized secondary mutation that resulted in more rapid growth without increased plasmid loss [51]: production of antihuman ovarian carcinoma×antihuman CD3 single chain antibody was increased 3‐fold in this double mutant compared with the starting host strain. This was essentially the same approach as that used previously [52], which was to isolate a strain that was more resistant to RPP‐induced stress.…”

Use of a Derivative of Escherichia coli BL21(DE3) for Efficient Production of Three Different Recombinant Proteins

“…Bacterial strain engineering based on the genetic selection of mutant strains has become one of the strategies for efficient production of recombinant proteins (25,26). In this work, the potential of BLRM(DE3), a host mutated from E. coli BLR(DE3) in our previous work, was further exploited for more efficient production of recombinant AhOC×AhCD3, Trx-hsBLyS, and Trx-hPTH.…”

“…Regulatory promoters for gene expression control are commonly used to balance the foreign gene expression level and the final cell density by separating the fermentation into two distinct stages: growth stage and production stage (19). Others strategies were also developed to overcome the negative effects of foreign gene overexpression on host cells, including reduction of plasmid copy number (20), chromosomal insertion of foreign genes (21), optimization of inducers and induction scheme (22,23), and bacterial strain engineering (24)(25)(26).…”