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Specific isolation by anhydrotrypsin‐agarose chromatography of a recombinant protein tagged with an affinity tail arginine at the C‐terminus
Author(s) -
Hirabayashi Jun,
Kasai Kenichi
Publication year - 1990
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.300030506
Subject(s) - affinity chromatography , agarose , recombinant dna , arginine , chemistry , biochemistry , chromatography , isolation (microbiology) , protein purification , microbiology and biotechnology , biology , amino acid , enzyme , bioinformatics , gene
A characteristic property of anhydrotrypsin, i.e., its ability to strongly bind C‐terminal arginine, proved to be useful as a tool for specific enrichment of a recombinant protein. An arginine tail was introduced at the C‐terminus, for example, of a human β‐galactoside‐binding lectin by site‐directed mutagenesis. The resulting mutant recombinant lectin, which retained sugar‐binding activity as high as the wild‐type lectin, became recognizable by anhydrotrypsin. It was adsorbed on an anhydrotrypsin‐agarose column and eluted with benzoylglycylarginine. The added arginine tail could be specifically removed by carboxypeptidase B. When E. coli lyzate containing the mutant lectin was applied to the column more than 10‐fold enrichment of the mutant lectin was attained. This procedure should be generally applicable and may be advantageous because the addition of a single arginine residue may have minimal effect on the structure and function of the target protein.