title: Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity.
abstract: 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.
authors.affiliation: British Bio-technology Ltd., Cowley, Oxford, U.K.
authors.forename: A L
authors.initials: AL
authors.lastname: Cook
authors.forename: P M
authors.initials: PM
authors.lastname: Kirwin
authors.forename: S
authors.initials: S
authors.lastname: Craig
authors.forename: L J
authors.initials: LJ
authors.lastname: Bawden
authors.forename: D R
authors.initials: DR
authors.lastname: Green
authors.forename: M J
authors.initials: MJ
authors.lastname: Price
authors.forename: S J
authors.initials: SJ
authors.lastname: Richardson
authors.forename: A
authors.initials: A
authors.lastname: Fallon
authors.forename: A H
authors.initials: AH
authors.lastname: Drummond
authors.forename: R M
authors.initials: RM
authors.lastname: Edwards
mesh_terms: 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
chemical_list: D007295:Inositol Phosphates; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; D010450:Endopeptidases