Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis.
In order to analyze the role of the pro-sequence in folding of the alkaline serine protease subtilisin, localized random mutagenesis using the polymerase chain reaction with Taq DNA polymerase was employed to obtain mutations in the pro-sequence which prevent production of active protease. The unique aspect of this procedure is that random mutations can be easily generated in vitro over large but defined regions of a specific gene. The method was applied to a 458-base pair fragment encompassing the coding region of the pro-sequence of subtilisin, a region of the protein which has been shown to be required for proper folding. Protease-deficient mutants containing a variety of amino acid substitutions were isolated with a frequency of 4.3%. From analysis of these mutants, four independent amino acid substitution mutations in the pro-sequence were identified. The present results demonstrate that polymerase chain reaction is an efficient and simple method for obtaining random mutations within a localized region of a given gene.
Lerner
C G
CG
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Rutgers, Piscataway 08554-5635.
Kobayashi
T
T
Inouye
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013381:Subtilisins / Q000096:biosynthesis / Q000235:genetics*
D004792:Enzyme Precursors; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; C057107:prosubtilisin; D013381:Subtilisins