title: Mutation in the Escherichia coli htpR locus results in stabilization of recombinant expression products that are susceptible to proteolytic degradation.
abstract: We compared the expression and degradation of three cloned malarial proteins in a pair of isogeneic strains of Escherichia coli that differed at the htpR locus. The htpR locus encodes an alternate sigma factor necessary for the transcription of heat shock promoters. Plasmodium sequences were cloned from polymerase chain reaction-amplified DNA initiated by oligonucleotide primers that were specific for the gene coding regions to be expressed. The amplified DNA was cloned and expressed in a vector that encodes a strong T7 promoter and translation--initiation signal. The total cell yield of two of the expressed proteins was found to be increased when synthesis occurred in a E. coli htpR mutant. Pulse--chase experiments showed that the increased protein yield correlated with a decrease in the degradation of the protein in the htpR strain. A two- to seven-fold increase in the half-life of the malaria proteins was observed in the E. coli htpR- background as compared to htpR+. We found no difference in survival of the E. coli K165 htpR mutant and isogeneic parent during thermal induction. Since the synthesis of the heat shock sigma factor did not significantly influence survival of E. coli and htpR expression results in increased degradation of foreign proteins, the E. coli htpR mutant was a valuable host strain for production of foreign proteins.
authors.affiliation: Department of Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.
authors.forename: L E
authors.initials: LE
authors.lastname: Lindler
authors.forename: J C
authors.initials: JC
authors.lastname: Anders
authors.forename: W E
authors.initials: WE
authors.lastname: Herman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010961:Plasmodium / Q000235:genetics* / Q000276:immunology; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics
chemical_list: D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases; D010447:Peptide Hydrolases