Analysis of recombinant protein toxicity in E. coli through a phage lambda-based genetic screening system.
The aspartic protease from the human immunodeficiency virus type 1 (HIV-1) is highly toxic to E. coli, thus impairing its yield in production processes. Proteolytic cleavage of essential cellular proteins is probably a major contributor to the bacteriocidal effect but this has not been proven. Through an adapted high-throughput lambda-based screening system, we have analyzed a set of HIV-1 protease mutants with distinguishable catalytic properties and we show that inactive enzymes are as toxic to E. coli cells as the wild-type enzyme. Together with additional data from directed molecular evolution approaches, these results indicate that the toxicity of the viral protease is not linked to its proteolytic activity. Our study also reveals that the lambda-based screening system is a robust new tool for the genetic analysis of highly toxic recombinant products in E. coli.
Fernández
Guerau
G
Fundació irsiCaixa, Universitat Autònoma de Barcelona, Hospital Universitari Germans Trias i Pujol, 08916, Badalona, Spain.
Vera
Andrea
A
Villaverde
Antonio
A
Martínez
Miguel Angel
MA
D010582:Bacteriophage lambda / Q000235:genetics*; D002470:Cell Survival / Q000502:physiology; D004926:Escherichia coli / Q000502:physiology*; D005820:Genetic Testing / Q000379:methods; D016333:HIV Protease / Q000235:genetics* / Q000378:metabolism*; D009152:Mutagenicity Tests / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D016333:HIV Protease