Extracellular Matrix Binding Properties of Recombinant Fibronectin Type II-like Modules of Human 72-kDa Gelatinase/Type IV Collagenase

The C‐terminal (haemopexin‐like) domain structure of human gelatinase A (MMP2): structural implications for its function

“…Fax: (49) (89) 8578 3516. E-mail: bode@biochem.mpg.de gelatinase B (MMP9) also bind collagen but do so using 3 fibronectin-related type-II modules, which are inserted into their catalytic domains [12][13][14][15][16]. They efficiently degrade native type-IV and -V collagens, all denatured collagens, elastin, and fibronectin [12,13,17,18]; none of these activities is lost upon removal of the CTD [4] (G. Murphy, unpubl, results).…”

Specific, High Affinity Binding of Tissue Inhibitor of Metalloproteinases-4 (TIMP-4) to the COOH-terminal Hemopexin-like Domain of Human Gelatinase A

“…Inclusion bodies from LE392 Escherichia coli expressing the recombinant proteins were solubilized in 6 M guanidine HCl, and recombinant protein was refolded in 0.1 M Na 2 B 4 O 7 , pH 10, followed by dialysis into either 20 mM or 100 mM Na 2 HPO 4 ⅐7H 2 0, NaH 2 PO 4 , pH 8.0, 0.5 M NaCl. rC domain and rCBD were purified using Zn 2ϩ -charged chelating Sepharose 6B (Pharmacia Biotech Inc.) affinity chromatography essentially as described previously (14,19). rCBD eluted from the Zn 2ϩ -chelate column was additionally applied to gelatin Sepharose 4B (Pharmacia) to select functionally folded CBD from the Zn 2ϩ -chelating Sepharose elute (14).…”

“…rC domain and rCBD were purified using Zn 2ϩ -charged chelating Sepharose 6B (Pharmacia Biotech Inc.) affinity chromatography essentially as described previously (14,19). rCBD eluted from the Zn 2ϩ -chelate column was additionally applied to gelatin Sepharose 4B (Pharmacia) to select functionally folded CBD from the Zn 2ϩ -chelating Sepharose elute (14). Protein yields were determined by the BCA assay (Pierce) and by spectroscopy after extinction coefficient determination (see below).…”

“…Briefly, the recombinant protein encoding the C domain and linker (Gly 417 -Cys 631 , exons 9 -13) and the CBD (Val 191 -Gln 364 , exons 5-7) of human gelatinase A were both expressed with a short NH 2 -terminal fusion tag that included an initiation methionine and a (His) 6 tag, using the expression vector pGYMX (14). Inclusion bodies from LE392 Escherichia coli expressing the recombinant proteins were solubilized in 6 M guanidine HCl, and recombinant protein was refolded in 0.1 M Na 2 B 4 O 7 , pH 10, followed by dialysis into either 20 mM or 100 mM Na 2 HPO 4 ⅐7H 2 0, NaH 2 PO 4 , pH 8.0, 0.5 M NaCl.…”

“…and CBD-Expression and purification of recombinant C domain (rC domain) and recombinant CBD (rCBD) was carried out essentially as described previously (14,19). Briefly, the recombinant protein encoding the C domain and linker (Gly 417 -Cys 631 , exons 9 -13) and the CBD (Val 191 -Gln 364 , exons 5-7) of human gelatinase A were both expressed with a short NH 2 -terminal fusion tag that included an initiation methionine and a (His) 6 tag, using the expression vector pGYMX (14).…”