Monitoring the cellular surface display of recombinant proteins by cysteine labeling and flow cytometry.
A general method is described that allows one to follow the surface display of recombinant proteins in Escherichia coli without having to use specific antibodies or enzymatic reactions. The method is based on cysteine-specific labeling through Michael addition to the double bond of maleimide and its derivatives, and takes advantage of the fact that naturally occurring surface proteins in E. coli contain no accessible cysteine residues. The method is easy to perform and could be simply applied to different analytic procedures including Western blot, spectral photometry, and flow cytometry. By using this new labeling method, single cells bearing a distinct protein at the surface could be selected by fluorescence-activated cell sorting. The data were obtained by using autodisplay, an efficient surface display system established for E. coli, but the method presented here represents rather a general solution for analyzing the surface display of recombinant proteins, independent of the cellular system used.
Jose
Joachim
J
Pharmazeutische und Medizinische Chemie Naturwissenschaftlich-Technische Fakultät III Universität des Saarlandes, P.O. Box 151150 66041 Saarbrücken, Germany. j.jose@mx.uni-saarland.de
Handel
Steffen
S
D000325:Adrenodoxin / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D002417:Cattle; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D019793:Fluorescein / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008301:Maleimides; D011993:Recombinant Fusion Proteins; D000325:Adrenodoxin; C043592:maleimide; D001710:Biotin; D003545:Cysteine; D019793:Fluorescein