Isolation of mutants of human immunodeficiency virus protease based on the toxicity of the enzyme in Escherichia coli.
The protease encoded by the pol gene of human immunodeficiency virus was expressed in Escherichia coli and found to be toxic to strain BL21(DE3). This toxicity provided a convenient selection for isolating mutants of the protease that are nontoxic and enzymatically inactive. This strong correlation between functional protease and toxicity resulted in rapid identification of several protease mutations, including mutations that exhibit temperature sensitivity. A total of 24 missense mutations and 7 nonsense mutations were identified. The described selection procedure may have wider applications for isolating mutants of other eukaryotic proteins that exhibit a toxic phenotype in E. coli.
Molecular Biology Section, Lederle Laboratories, American Cyanamid Company, Pearl River, NY 10965.
E Z
EZ
Baum
G A
GA
Bebernitz
Y
Y
Gluzman
D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D015684:Gene Products, pol / Q000235:genetics*; D006678:HIV / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010447:Peptide Hydrolases / Q000235:genetics*; D010957:Plasmids
D003062:Codon; D015684:Gene Products, pol; D015345:Oligonucleotide Probes; D010447:Peptide Hydrolases