Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus

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• 作者：B. Sauerzapfe ; D.-J. Namdjou ; T. Schumacher ; N. Linden ; K. Kř ; enek ; V. Kř ; en ; L. Elling
• 关键词：Human β ; 1 ; 4-galactosyltransferase ; Fusion protein ; Lipase propeptide ; Staphylococcus hyicus ; Hydrophobic acceptor substrates
• 刊名：Journal of Molecular Catalysis B: Enzymatic
• 出版年：2008
• 出版时间：1 February 2008
• 年：2008
• 卷：50
• 期：2-4
• 页码：128-140
• 全文大小：980 K

文摘

The question whether proteins fused to β1,4galactosyltransferase (β4GalT-1) influence the biocatalytic properties of the glycosyltransferase has not been addressed so far. In the present study we have chosen a novel approach to express the gene encoding for human β4GalT-1 from placenta in an N-terminal fusion with the pre-propeptide of the lipase from Staphylococcus hyicus. The pre-propeptide provides a secretion signal in Escherichia coli and was reported to protect fused proteins against proteolytic degradation. Expression of the fusion protein was challenged with an almost full-length version of human β4GalT-1 including parts of the signal anchor and the stem region (propeptide-natβ4GalT-1) and the full catalytic domain (His₆propeptide-catβ4GalT-1), respectively. We demonstrate that the fusion protein in propeptide-natβ4GalT-1 is cleaved off during purification using immobilized metal ion chromatography IMAC, most probably catalyzed by the immobilized Zn²⁺ ions. Cleavage can be avoided by deletion of five C-terminal amino acids of the propeptide and the stem region yielding His₆propeptide-catβ4GalT-1. Kinetic data reveal that both enzyme constructs possess specific biocatalytic characteristics when compared to a recombinant luminal β4GalT-1 construct. The catalytic efficiency towards more hydrophobic acceptor glycosides, e.g. benzyl 2-acetamido-2-deoxy-β-d-glucopyranoside and p-nitro phenyl 2-acetamido-2-deoxy-β-d-glucopyranoside, and the N-glycans of IgG from rat is significantly increased. In summary, the His₆propeptide-catβ4GalT-1 is very useful for biocatalytic applications involving hydrophobic acceptor glycosides and the propeptide-natβ4GalT-1 is able to glycosylate glycoproteins in an efficient way.