A method of screening for mutant proteins containing cysteine residues using fluorescein-5-maleimide.
A method of screening transformed bacterial colonies for introduction of a cysteine residue into an overexpressed protein is described. After treating SDS extracts of induced bacterial cells with fluorescein-5-maleimide, the proteins containing cysteine were visible on SDS-PAGE gels under ultraviolet light as fluorescent bands. If the wild-type protein contains no endogenous cysteine residues, then mutant proteins containing cysteine may be easily identified by their fluorescence. In addition, a shift in electrophoretic mobility of modified proteins was observed, with mutant proteins containing cysteine at more than one site exhibiting incremental decreases in electrophoretic mobility. This effect permits the detection of cysteine mutations even when endogenous cysteines are present. The described method allows the rapid screening of a large number of transformants.
McLachlin
D T
DT
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
Dunn
S D
SD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003545:Cysteine / Q000032:analysis*; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005452:Fluoresceins / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D005452:Fluoresceins; C037041:fluorescein 5-maleimide; D004247:DNA; D004720:Endonucleases; D003545:Cysteine