Expression and purification of His-tagged beta-1,4-galactosyltransferase in yeast and in COS cells.
His6-tag technology has been introduced for easy purification of recombinant proteins expressed in Escherichia coli. Aiming at extending this technology to purification of glycoproteins expressed in Saccharomyces cerevisiae or in animal cells, respectively, we adapted this protocol to recombinant soluble beta-1,4-galactosyltransferase (rgal-T). A His6-tag was introduced to the N-terminus of the protein (hisGal-T). The Histagged enzyme expressed in yeast S. cerevisiae was 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.
Borsig
L
L
Institute of Physiology, Zurich, Switzerland.
Berger
E G
EG
Malissard
M
M
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000201:enzymology*; D002847:Chromatography, Agarose; D004800:Enzymes, Immobilized; D015870:Gene Expression; D006031:Glycosylation; D006639:Histidine; D016297:Mutagenesis, Site-Directed; D009237:N-Acetyllactosamine Synthase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012995:Solubility; D014162:Transfection / Q000235:genetics
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D009237:N-Acetyllactosamine Synthase