Polylinker-Encoded Peptides Can Confer Toxicity to Recombinant Proteins Produced in Escherichia coli

Abstract

Three DNA segments encoding fragments of the p60 capsid protein of rabbit haemorrhagic disease virus (RHDV) have been cloned and expressed in Escherichia coli. The cDNAs were placed under the control of the T7 promoter in a pET-derived expression vector designed to produce C-terminal histidine tail fusions. By using different cloning strategies, cell toxicity exhibited by some of the expressed products was dramatically affected, coincident with minor modifications in the carboxy terminus sequences of the recombinant proteins. In particular, the presence of a nonhydrophobic peptide encoded by polylinker sequences promotes cell death and a reduced yield after induction of gene expression, whereas a histidine tail has no detectable effect. These data point out the critical role that needless peptide tails, accidentally introduced into recombinant proteins by nonrelevant DNA stretches, can have on protein expression and final yield of a production process.

Tolerance of Escherichia coli β‐galactosidase C‐terminus to different‐sized fusions

“…Since no significant differences between growth of cultures producing LAC10, LAC110 and LAC174 were detected, we can argue that there might be other factors, different from molecular mass, affecting growth of MC1061/pJLACVP1 cells. In fact, some specific amino acid sequences are known to cause toxicity on the producing cells (Couto and Friedberg, 1989;Tsai et al, 1989;Viaplana and Villaverde, 1996). Production of complete, unfused VP1 protein was reported to induce severe symptoms of plasmid instability and toxicity, even under non-inducing conditions (Vidal et al, 1991).…”

Metabolic adaptation of Escherichia coli during temperature‐induced recombinant protein production: 2. Redirection of metabolic fluxes

“…The production of a recombinant protein can result in a variety of cellular stress responses, which can be reflected by reduced growth rates (e.g., Viaplana and Villaverde, 1996), ribosome destruction (Dong et al, 1995), stress protein synthesis (e.g., Hoffmann and Rinas, 2000;Parsell and Sauer, 1989;Ramirez and Bentley, 1993), and alterations of central carbon metabolism (e.g., George et al, 1992). For quantitative evaluation of the metabolic perturbation, the analysis of metabolic fluxes has emerged as an important tool in the field of metabolic engineering (Vallino and Stephanopoulos, 1990).…”

Interplay of SOS induction, recombinant gene expression, and multimerization of plasmid vectors in Escherichia coli

“…Although the genetics and physiology of E. coli are well characterized, the cellular mechanism of the cell damage caused by heterologous proteins remains unclear. To date, it has been reported that a strong SOS response is induced with the expression of diverse heterologous proteins, including enzymes and nonenzymatic proteins/ peptides (Aris et al, 1998;Bedouelle et al, 1990;Benito et al, 1995;Laity et al, 1993;Pham and Coleman, 1985;Sisk et al, 1992;Viaplana and Villaverde, 1996;Vidal et al, 1991;Colasanti and Denhardt, 1985). From the fact that the SOS response is induced irrespective of the biological function of the expressed recombinant proteins, Figure 6.…”

“…They concluded that the observed SOS response could be related to either the metabolic burden or the toxicity associated with the production of recombinant proteins. For other products, the presence of particular amino acid sequences could be responsible for their toxicity (Sisk et al, 1992;Viaplana and Villaverde, 1996;Vidal et al, 1991), and their removal rendered nontoxic products and increased the yield (Sisk et al, 1992;Vidal et al, 1991). It seems that the SOS response is induced in recombinant E. coli regardless of the biological properties of synthesized foreign proteins/peptides; the reason why the SOS-inducing events happen along with recombinant gene expression still remains unclear.…”