Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity.
Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen. A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB. Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32. Substitution of this arginine residue, or arginine-28 [a potential KEX2 (lysine-arginine endopeptidase) cleavage site], prevents or reduces cleavage of PDGF-BB respectively. These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF-BB. These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson [(1990) Mol. Cell Biol. 10, 5496-5501] suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding.
Cook
A L
AL
British Bio-technology Ltd., Cowley, Oxford, U.K.
Kirwin
P M
PM
Craig
S
S
Bawden
L J
LJ
Green
D R
DR
Price
M J
MJ
Richardson
S J
SJ
Fallon
A
A
Drummond
A H
AH
Edwards
R M
RM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D007295:Inositol Phosphates; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D010450:Endopeptidases