Arginine-tail method, an affinity tag procedure utilizing anhydrotrypsin agarose.
The arginine-tail method is a recently developed affinity tag procedure utilizing immobilized anhydrotrypsin for specific enrichment of a recombinant protein. Three model proteins (originally human beta-galactoside-binding lectin with a relative sub-unit molecular mass of 14,000) were prepared by mutagenesis, each of which has a tail of either Arg, Gly-Arg, or Gly-Gly-Arg at the C-terminus. All of them retained their original sugar-binding activity and antigenicity, and became recognizable by anhydrotrypsin. They were adsorbed on an anhydrotrypsin-agarose column at pH 5 after treatment with 4 M urea or 10 mM HCl to expose the C-terminal tails. The adsorbed arginine-tailed lectins were eluted either specifically with benzoylglycylarginine (Bz-Gly-Arg) or with 5 mM HCl. Added arginine was removed by carboxypeptidase B, but very slowly. When urea-denatured Escherichia coli lysate containing one of the arginine-tailed lectins was applied to the column, the lectin was adsorbed together with some host proteins, which are expected to have arginine or lysine at their C-termini. However, more than a ten-fold enrichment was attained by this procedure. The described method would be useful for purifying various recombinant proteins, even those which are inactive as a result of mutagenesis. The smallest tag arginine at the C-terminus should have a minimum effect on both the structure and function of a target protein.
Hirabayashi
J
J
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Kasai
K
K
D000327:Adsorption; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D043424:Carboxypeptidase B; D002268:Carboxypeptidases; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D015870:Gene Expression; D007202:Indicators and Reagents; D037102:Lectins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D012685:Sepharose; D014357:Trypsin / Q000737:chemistry*
D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D037102:Lectins; C011661:anhydrotrypsin; D012685:Sepharose; D001120:Arginine; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin