Expression and purification of soluble and inactive mutant forms of membrane type 1 matrix metalloproteinase.
Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteinase and a cell-surface receptor and activator of gelatinase A in normal and neoplastic cells. We have expressed and purified a soluble deletion mutant of MT1-MMP lacking the transmembrane and cytoplasmic domains and an inactive mutant of the soluble MT1-MMP, where the active-site glutamic acid(240) was substituted by alanine (E240A). A baculovirus transfer vector coding for amino acids 21-539 of MT1-MMP (DeltaTM) and a similar vector coding for the mutation (E240ADeltaTM) were constructed for expression in insect cells. Both DeltaTM and E240ADeltaTM were secreted to the culture medium of infected High Five insect cells. They were then purified by cation-exchange followed by gel-filtration chromatography. DeltaTM was able to cleave denatured type I collagen and fibronectin and activate MMP-2/gelatinase-A, while E240ADeltaTM had only low proteolytic activity against denatured collagen I. The current expression and purification protocol should prove useful for the production of large amounts of enzymatically active soluble MT1-MMP.
Valtanen
H
H
Department of Virology, The Haartman Institute, Helsinki, FIN-00014, Finland.
Lehti
K
K
Lohi
J
J
Keski-Oja
J
J
D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000737:chemistry; D005780:Gelatin / Q000737:chemistry; D005822:Genetic Vectors; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion; D018411:Spodoptera / Q000166:cytology
D005353:Fibronectins; D011994:Recombinant Proteins; D005780:Gelatin; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2