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 the 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 greater percentage of the expressed fusion protein was soluble, monomeric, and undegraded when the growth temperature was lowered, the growth medium was supplemented with zinc, and metal chelators were omitted from the lysis buffers. Similar effects were observed when we tested the effects of lower growth temperature and zinc supplementation on another 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 zinc 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 requirement for zinc necessary for binding to aggrecan. Factor Xa cleavage of MBP/LP expressed in the presence of zinc yielded much more intact LP product than cleavage of MBP/LP expressed without zinc. These data indicate a structural role of zinc that allows MBP/LP to fold in a manner such that it is resistant to proteolytic degradation.
Varelas
J B
JB
Department of Medicine, Case Western Reserve University, Cleveland, Ohio, 44106-4946, USA. jbv2@po.cwru.edu
Roy
C
C
Hering
T M
TM
D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D006820:Hyaluronic Acid / Q000378:metabolism; D037241:Mannose-Binding Lectins; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D015032:Zinc / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000099249:Proteoglycan Link Protein
D002352:Carrier Proteins; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D015951:Factor Xa; D006820:Hyaluronic Acid; D037241:Mannose-Binding Lectins; D011506:Proteins; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D015032:Zinc; D000099249:Proteoglycan Link Protein