Screening for soluble expression constructs using cell-free protein synthesis.
The SH2 domain of STAT6 was chosen to test the in vitro protein synthesis as a screening tool. Goal of the screening was to obtain constructs which produce soluble protein in E. coli. The expression of 70 different constructs using an E. coli based cell-free system revealed two constructs, which give partly soluble protein. The introduction of two mutations, which had been suggested by a structural based alignment of 20 different SH2 domains lead to increased solubility. The expression of both constructs in E. coli followed by an affinity and size exclusion chromatography resulted in milligram quantities of highly purified protein.
Lamla
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Department of Lead Discovery, Boehringer Ingelheim Pharma GmbH and Co. KG, Birkendorfer Strasse 65, D-88397 Biberach/Riss, Germany. thorsten.lamla@bc.boehringer-ingelheim.com
Hoerer
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Bauer
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D000818:Animals; D002417:Cattle; D002474:Cell-Free System / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051379:Mice; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D050800:STAT6 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human