Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris.
A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).
Faculty of Chemistry/Biochemistry I, University of Bielefeld, Universitätsstrasse 25, Bielefeld, D-33615, Germany.
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Roderfeld
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FH
Büttner
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Bartnik
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H
Tschesche
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D020781:Matrix Metalloproteinase 1