"Cystope tagging" for labeling and detection of recombinant protein expression.
A labeling and detection method, based on the addition of a single cysteine residue at the C terminus of a recombinant protein and the subsequent sulfhydryl-specific Michael addition to the double bond of maleimide and its derivatives, was developed. The method was named "cystope tagging." Sorbit dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase family of proteins that contains three inherent cysteines, was used as a model recombinant protein. By labeling with fluorescein-maleimide, it was demonstrated that only the single accessory cysteine is accessible under nonreducing conditions. After the addition of beta-mercaptoethanol, the inherent cysteines of SDH were also detectable by coupling to fluorescein-maleimide. The data were obtained using Autodisplay, an efficient surface expression system in Escherichia coli, but the method presented in this article represents a rather general solution for analyzing the expression of recombinant proteins, irrespective of the expression system used. The authors conclude that cystope tagging is an interesting alternative to other tagging methods applied in recombinant protein techniques.
Jose
Joachim
J
Pharmaceutical and Medicinal Chemistry, University of the Saarland, P.O. Box 151150, Im Stadtwald, Geb.32, 66041 Saarbrücken, Germany. joachim.jose@uni-duesseldorf.de
von Schwichow
Steffen
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004274:DNA, Recombinant; D007064:L-Iditol 2-Dehydrogenase / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000201:enzymology
D017931:DNA Primers; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D007064:L-Iditol 2-Dehydrogenase