Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins in Escherichia coli.
The major leftward early promoter of phage lambda pL, has frequently been used to drive expression of heterologous genes in Escherichia coli. pL is typically maintained fully repressed by the lambda cl protein. When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences. One method of derepressing pL involves exposing cells to nalidixic acid, which results in the "activation" of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl. Activated RecA also mediates the cleavage of the E. coli LexA protein, resulting in induction of the SOS regulon (at least 15 E. coli genes, including recA). We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions. One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind-) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage. These mutations were introduced into a strain carrying a cl+ defective lysogen. Synthesis of two heterologous proteins, human alpha 1-antitrypsin and a fusion protein partially derived from the Plasmodium falciparum circumsporozoite surface antigen, was examined in the wild-type and mutant strains. The maximum alpha-1 antitrypsin concentration achieved was improved by 50% when the recA o strain was used rather than the wild-type; however, only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein were observed. Use of the lexAind- strain resulted in a decrease in the maximum concentration attained for both heterologous products.
Keller
J
J
Department of Biological Process Sciences, Smith Kline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Gerber
R
R
Del Tito
B J
BJ
Vannicolo
D
D
Arcuri
E J
EJ
D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation; D009154:Mutation; D009268:Nalidixic Acid / Q000494:pharmacology*; D010963:Plasmodium falciparum; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013014:SOS Response, Genetics; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000235:genetics
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; C041595:circumsporozoite protein, Protozoan; D009268:Nalidixic Acid