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 ultraviole...

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Bibliographic Details
Published inProtein expression and purification Vol. 7; no. 3; pp. 275 - 280
Main Authors McLachlin, Derek T., Dunn, Stanley D.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 01.05.1996
Subjects
Amino Acid Sequence
Bacterial Proteins - biosynthesis
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Base Sequence
Cysteine - analysis
DNA - chemical synthesis
DNA - genetics
Electrophoresis, Polyacrylamide Gel
Endonucleases - genetics
Escherichia coli - genetics
Fluoresceins - chemistry
Molecular Sequence Data
Plasmids - genetics
Spectrometry, Fluorescence
Transformation, Genetic
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Summary: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.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
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ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1996.0039