STREP-TAG-II AFFINITY PURIFICATION - AN APPROACH TO STUDY INTERMEDIATES OF METALLOENZYME BIOSYNTHESIS

Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are sy nthesized starting from the apoprotein via several intermediates by th e action of accessory proteins. The isolation and biochemical characte rization of such intermediates is hampered by their low abundance and their lability. Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Seph arose matrix. The tag was fused to the large subunit of [NiFe] hydroge nase 3 (HycE) of Escherichia coli. No significant influence of the aff inity peptide on maturation or activity of the protein was observed wh en the modified gene was integrated into the chromosome by homologous recombination. A tagged nickel-free precursor form of HycE bound quant itatively to a recombinant StrepTactin Sepharose column. More than 90% pure subunit could be obtained after elution with desthiobiotin. The procedure was shown to be more efficient than purification by immobili zed metal affinity chromatography using a N-terminal His-tag. General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed. (C) 1998 Academic Press.