Arginine-tail method, an affinity tag procedure utilizing anhydrotrypsin agarose.

Abstract

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.