Purification and Analysis of Proteinase-resistant Mutants of Recombinant Platelet-derived Growth Factor-BB Exhibiting Improved Biological Activity

Overview

Journal Biochem J

Specialty Biochemistry

Date 1992 Jan 1

PMID 1731768

Citations 3

Authors

A L Cook

P M Kirwin

S Craig

L J Bawden

D R Green

M J Price

S J Richardson

A Fallon

A H Drummond

R M Edwards

Affiliations

Soon will be listed here.

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.

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Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

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References

Seifert R, Hart C, Phillips P, Forstrom J, Ross R, Murray M . Two different subunits associate to create isoform-specific platelet-derived growth factor receptors. J Biol Chem. 1989; 264(15):8771-8. View

Hoppe J, Weich H, Eichner W . Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli. Biochemistry. 1989; 28(7):2956-60. DOI: 10.1021/bi00433a032. View

Settineri C, Medzihradszky K, Masiarz F, Burlingame A, Chu C . Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis. Biomed Environ Mass Spectrom. 1990; 19(11):665-76. DOI: 10.1002/bms.1200191106. View

Craig S, Clements J, Cook A, Dryden D, Green D, Heremans K . Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae. Biochem J. 1992; 281 ( Pt 1):67-72. PMC: 1130641. DOI: 10.1042/bj2810067. View

Hammacher A, Nister M, Westermark B, Heldin C . A human glioma cell line secretes three structurally and functionally different dimeric forms of platelet-derived growth factor. Eur J Biochem. 1988; 176(1):179-86. DOI: 10.1111/j.1432-1033.1988.tb14266.x. View

Ross R, Raines E, Bowen-Pope D . The biology of platelet-derived growth factor. Cell. 1986; 46(2):155-69. DOI: 10.1016/0092-8674(86)90733-6. View

Ostman A, Rall L, Hammacher A, Wormstead M, Coit D, Valenzuela P . Synthesis and assembly of a functionally active recombinant platelet-derived growth factor AB heterodimer. J Biol Chem. 1988; 263(31):16202-8. View

Hammacher A, Mellstrom K, Heldin C, Westermark B . Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer. EMBO J. 1989; 8(9):2489-95. PMC: 401239. DOI: 10.1002/j.1460-2075.1989.tb08385.x. View

Heldin C, Ernlund A, Rorsman C, Ronnstrand L . Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation. J Biol Chem. 1989; 264(15):8905-12. View

10.

Mizuno K, Nakamura T, Ohshima T, Tanaka S, Matsuo H . Characterization of KEX2-encoded endopeptidase from yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1989; 159(1):305-11. DOI: 10.1016/0006-291x(89)92438-8. View

11.

Ostman A, Backstrom G, Fong N, Betsholtz C, Wernstedt C, Hellman U . Expression of three recombinant homodimeric isoforms of PDGF in Saccharomyces cerevisiae: evidence for difference in receptor binding and functional activities. Growth Factors. 1989; 1(3):271-81. DOI: 10.3109/08977198908998003. View

12.

Nister M, Hammacher A, Mellstrom K, Siegbahn A, Ronnstrand L, Westermark B . A glioma-derived PDGF A chain homodimer has different functional activities from a PDGF AB heterodimer purified from human platelets. Cell. 1988; 52(6):791-9. DOI: 10.1016/0092-8674(88)90421-7. View

13.

Mizuno K, Nakamura T, Ohshima T, Tanaka S, Matsuo H . Yeast KEX2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochem Biophys Res Commun. 1988; 156(1):246-54. DOI: 10.1016/s0006-291x(88)80832-5. View

14.

Sauer M, Donoghue D . Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor. Mol Cell Biol. 1988; 8(3):1011-8. PMC: 363243. DOI: 10.1128/mcb.8.3.1011-1018.1988. View

15.

Eichner W, Jager V, Herbst D, Hauser H, Hoppe J . Large-scale preparation of recombinant platelet-derived growth factor AA secreted from recombinant baby hamster kidney cells. Eur J Biochem. 1989; 185(1):135-40. DOI: 10.1111/j.1432-1033.1989.tb15094.x. View

16.

West Jr R, Yocum R, Ptashne M . Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Mol Cell Biol. 1984; 4(11):2467-78. PMC: 369078. DOI: 10.1128/mcb.4.11.2467-2478.1984. View

17.

Kunkel T, Roberts J, Zakour R . Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987; 154:367-82. DOI: 10.1016/0076-6879(87)54085-x. View

18.

Stroobant P, Waterfield M . Purification and properties of porcine platelet-derived growth factor. EMBO J. 1984; 3(12):2963-7. PMC: 557798. DOI: 10.1002/j.1460-2075.1984.tb02241.x. View

19.

Sauer M, Hannink M, Donoghue D . Deletions in the N-terminal coding region of the v-sis gene: determination of the minimal transforming region. J Virol. 1986; 59(2):292-300. PMC: 253078. DOI: 10.1128/JVI.59.2.292-300.1986. View

20.

Heldin C, Johnsson A, Wennergren S, Wernstedt C, Betsholtz C, Westermark B . A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains. Nature. 1986; 319(6053):511-4. DOI: 10.1038/319511a0. View