MUTATION OF CYS(672) ALLOWS RECOMBINANT EXPRESSION OF ACTIVATIBLE MACROPHAGE-STIMULATING PROTEIN

We readily produced recombinant pro-macrophage stimulating protein in a mammalian expression system, but it was only weakly active after pro teolytic activation. Active macrophage stimulating protein is a disulf ide-bonded heterodimer, but in our hands, the subunits of recombinant macrophage stimulating protein were mostly not disulfide bonded, Molec ular modeling of the serine proteinase domain of macrophage stimulatin g protein based on homology to human trypsin suggested that macrophage stimulating protein, but not plasminogen or hepatocyte growth factor, has a Cys residue (672) in close proximity to the Cys residue (578) t hat forms the intersubunit disulfide link with the other subunit. We h ypothesized that Cys(672) might interfere with intersubunit disulfide formation by forming an intrasubunit disulfide with Cys(578) and there fore mutated Cys(672) to Ala. After kallikrein activation, the subunit s of Cys(672) --> Ala macrophage stimulating protein were fully disulf ide linked, and the mutant macrophage stimulating protein had 10-20 fo ld higher specific activity than the wild type recombinant macrophage stimulating protein.