A STRUCTURAL REQUIREMENT OF ZINC FOR THE FOLDING OF RECOMBINANT LINK PROTEIN

We have cloned and ligated a full-length bovine link protein (LP) in t he pMAL-c2 vector and overexpressed it in fusion with maltose-binding protein (MBP) in Escherichia coli. We have demonstrated dose-dependent binding of MBP/LP to biotinylated hyaluronan in a dot blot assay, A g reater percentage of the expressed fusion protein was soluble, monomer ic, and undegraded when the growth temperature was lowered, the growth medium was supplemented with zinc, and metal chelators were omitted f rom the lysis buffers, Similar effects were observed when we tested th e effects of lower growth temperature and zinc supplementation on anot her construct consisting of MBP in fusion with the first proteoglycan tandem repeat of LP, Our results suggest zinc may be necessary for the folding and disulfide bond formation of recombinant LP, In addition, a greater amount of monomeric MBP/LP produced at 27 degrees C with zin c supplementation bound to biotinylated hyaluronic acid-binding region of aggrecan than MBP/LP produced at 27 or 37 degrees C without zinc, This suggests that recombinant LP may have a conformational requiremen t for zinc necessary for binding to aggrecan, Factor Xa cleavage of MB P/LP expressed in the presence of zinc yielded much more intact LP pro duct than cleavage of MBP/LP expressed without zinc, These data indica te a structural role of zinc that allows MBF/LP to fold in a manner su ch that it is resistant to proteolytic degradation. (C) 1997 Academic Press.