Abstract
His6-tag technology has been introduced for easy purification of recombinant proteins expressed inEscherichia coli.Aiming at extending this technology to purification of glycoproteins expressed inSaccharomyces cerevisiaeor in animal cells, respectively, we adapted this protocol to recombinant soluble β-1,4-galactosyltransferase (rgal-T). A His6-tag was introduced to the N-terminus of the protein (hisGal-T). The His-tagged enzyme expressed in yeastS. cerevisiaewas enzymically active but could not be purified from the cell extract by virtue of the His6-tag. Binding efficiency of hisGal-T was found to be impaired by a bulky N-glycan close to the His-tag. Removal of the unique site of N-glycosylation using site-directed mutagenesis restored binding of hisGal-T to the Ni-NTA resin. In comparison N-glycosylated hisGal-T transiently expressed in COS cells was secreted as a soluble active enzyme and could be purified in one single step by virtue of the His6-tag.
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