Mutation of Cys672 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 proteolytic activation. Active macrophage stimulating protein is a disulfide-bonded heterodimer, but in our hands, the subunits of recombinant macrophage stimulating protein were mostly not disulfide bonded. Molecular modeling of the serine proteinase domain of macrophage stimulating 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) that forms the intersubunit disulfide link with the other subunit. We hypothesized that Cys672 might interfere with intersubunit disulfide formation by forming an intrasubunit disulfide with Cys578 and therefore mutated Cys672 to Ala. After kallikrein activation, the subunits of Cys672 --> Ala macrophage stimulating protein were fully disulfide linked, and the mutant macrophage stimulating protein had 10-20-fold higher specific activity than the wild type recombinant macrophage stimulating protein.
Wahl
R C
RC
Amgen, Inc., Thousand Oaks, California 91320-1789, USA. rwahl@amgen.com
Costigan
V J
VJ
Batac
J P
JP
Chen
K
K
Cam
L
L
Courchesne
P L
PL
Patterson
S D
SD
Zhang
K
K
Pacifici
R E
RE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics*; D006133:Growth Substances / Q000235:genetics*; D017228:Hepatocyte Growth Factor; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D006133:Growth Substances; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor; D003545:Cysteine