Abstract
Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is used to treat full-thickness diabetic ulcers and is being investigated for use in other chronic ulcers, non-healing wounds, and periodontal defects. A simple, novel method for expression and purification of rhPDGF-BB from Escherichia coli is now described. This method produces the dimeric protein in high yield (10–12 mg/g wet cell mass) and with a purity >95%. rhPDGF-BB was exclusively found in inclusion bodies (IBs) representing approx. 30% of the total cell proteins. The IBs were extracted and the monomer purified by RP-HPLC. The purified rhPDGF-B monomer was then refolded using Tris buffer and subsequently dimerized to produce biologically active rhPDGF-BB. This product was composed of two polypeptide chains, each approx. 12 kDa. The final product exhibited specific activity in a fibroblast proliferation assay indistinguishable from that of the WHO reference standard.
References
Alexander DM, Hesson T, Mannarino A et al (1992) Isolation and purification of a biologically active human platelet-derived growth factor BB expressed in Escherichia coli. Protein Expr Purif 3:204–211
Claesson-Welsh L, Heldin CH (1989) Platelet-derived growth factor. Three isoforms that bind to two distinct cell surface receptors. Acta Oncol 28:331–334
Cook AL, Kirwin PM, Craig S et al (1992) Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity. Biochem J 281(Pt 1):57–65
Deuel T, Huang J (1984) Platelet-derived growth factor. Structure, function, and roles in normal and transformed cells. J Clin Invest 74:669–676
Giese N, May-Siroff M, LaRochelle WJ et al (1989) Expression and purification of biologically active v-sis/platelet-derived growth factor B protein by using a baculovirus vector system. J Virol 63:3080–3086
Harrison-Balestra C, Eaglstein WH, Falabela AF et al (2002) Recombinant human platelet-derived growth factor for refractory nondiabetic ulcers: a retrospective series. Dermatol Surg 28:755–759
Heldin C-F, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316
Hom DB, Manivel JC (2003) Promoting healing with recombinant human platelet-derived growth factor-BB in a previously irradiated problem wound. Laryngoscope 113:1566–1571
Hoppe J, Weich HA, Eichner W (1989) Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli. Biochemistry 28:2956–2960
Jacobson A (1987) Purification and fractionation of poly(A)+ RNA. Methods Enzymol 152:254–261
Johnsson A, Heldin CH, Westermark B et al (1982) Platelet-derived growth factor: identification of constituent polypeptide chains. Biochem Biophys Res Commun 104:66–74
Kallianinen LK, Hirshberg J, Marchant B et al (2000) Role of platelet-derived growth factor as an adjunct to surgery in the management of pressure ulcers. Plast Reconstr Surg 106:1243–1248
Kenney WC, Haniu M, Herman AC et al (1994) Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds. J Biol Chem 269:12351–12359
Mandracchia VJ, Sanders SM, Frerichs JA (2001) The use of becaplermin (rhPDGF-BB) gel for chronic nonhealing ulcers. A retrospective analysis. Clin Podiatr Med Surg 18:189–209
Meyer-Ingold W, Eichner W (1995) Platelet-derived growth factor. Cell Biol Int 19:389–398
Mohammadian-Mosaabadi J, Naderi-Manesh H, Maghsoudi N et al (2006) Improving purification of recombinant human interferon γ expressed in Escherichia coli; effect of removal of impurity on the process yield. Protein Expr Purif 51:147–156
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxic assays. J Immunol Methods 65:55–63
Nevins M, Giannobile WV, McGuire MK et al (2005) Platelet-derived growth factor stimulates bone fill and rate of attachment level gain: results of a large multicenter randomized controlled trial. J Periodontol 76:2205–2215
Oefner C, D’Arcy A, Winkler FK et al (1992) Crystal structure of human platelet-derived growth factor BB. Embo J 11:3921–3926
OMJ Pharmaceuticals (1999) REGRANEX Gel 0.01% (Ortho-McNeil) (becaplermin). San German, Puerto Rico
Pierce GF, Mustoe TA, Altrock BW et al (1991) Role of platelet-derived growth factor in wound healing. J Cell Biochem 45:319–326
Raines EW, Ross R (1985) Purification of human platelet-derived growth factor. Methods Enzymol 109:749–773
Ross R, Vogel A (1978) The platelet-derived growth factor. Cell 14:203–210
Senet P (2004) [Becaplermin gel (Regranex gel)]. Ann Dermatol Venereol 131:351–358
Watson E, Kenney WC (1992) Multiple peak formation from reversed-phase liquid chromatography of recombinant human platelet-derived growth factor. J Chromatogr 606:165–170
Wieman TJ (1998) Clinical efficacy of becaplermin (rhPDGF-BB) gel. Becaplermin Gel Studies Group. Am J Surg 176:74S–79S
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This work was supported by NIH Grant HL070068 (R.C.R.).
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Karumuri, N.N., Gangireddy, S.R., Narala, V.R. et al. Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB. Biotechnol Lett 29, 1333–1339 (2007). https://doi.org/10.1007/s10529-007-9411-9
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DOI: https://doi.org/10.1007/s10529-007-9411-9