Matrilysin: expression, purification, and characterization.
The expression vector pGEX-2T under the control of the IPTG-inducible tac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin, HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.
Soler
D
D
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nomizu
T
T
Brown
W E
WE
Shibata
Y
Y
Auld
D S
DS
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005823:Genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007700:Kinetics; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D015032:Zinc / Q000378:metabolism
D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7; D015032:Zinc