Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity

Cook, A; Kirwin, P M; Craig, Stewart; Bawden, Lindsay J.; Green, D R; Price, M; Richardson, Samantha J.; Fallon, A.; Drummond, Alan H.; Edwards, Richard M.

doi:10.1042/bj2810057

Cited by 12 publications

(3 citation statements)

References 58 publications

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“…This strategy has been applied to platelet-derived growth factor (PDGF)-BB [41]. Site directed mutagenesis was used to generate Arg32Pro and Arg28Ser PDGF-BB mutants which exhibited 5-fold increased expression levels of PDGF-BB, and also demonstrated a 2–3 fold improvement in mitogenic potency compared to wild-type PDGF-BB [41]. Similar protein engineering strategies can be applied to other growth factor families to address manufacturing challenges and would serve to effectively reduce cost and time to production.…”

Section: Protein Engineering Technologies Applied To Growth Factorsmentioning

confidence: 99%

Engineering growth factors for regenerative medicine applications

et al. 2016

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Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.

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Section: Protein Engineering Technologies Applied To Growth Factorsmentioning

confidence: 99%

Engineering growth factors for regenerative medicine applications

et al. 2016

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“…Cook et al (1992) used a yeast expression system and purified the protein using two-step size exclusion chromatography. However, expression was low and transfer into an intermediate buffer between the two chromatography steps was required to achieve satisfactory yields.…”

Section: Discussionmentioning

confidence: 99%

Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB

et al. 2007

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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.

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“…The rhPDGF-BB has been approved by FDA as the treatment for diabetic foot ulcer and self-bone grafting [1] . It has been produced in a variety of heterologous systems including Escherichia coli [2][3][4] , Chinese hamster ovary cells [5] , Saccharomyces cerevisiae [6,7] , baculovirus [8] , vaccinia viruses [9] , mushroom [10] , plant [11] and Pichia pastoris [12] . Babavalian et al [12] reported a high efficiency of 30 mg/L in Pichia pink, a P. pastoris mutant cell, without optimization.…”

Section: Introductionmentioning

confidence: 99%

Storage of the Recombinant Protein hPDGF-BB in the Culture of Pichia pastoris

Pham¹,

Quoc²,

Le³

et al. 2018

IJLSSR

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Human platelet-derived growth factor-BB (hPDGF-BB), a proliferation factor, has been successfully manufactured and approved by FDA as the treatment for diabetic foot ulcer and self bone grafting. There have been no reports on the storage of the recombinant hPDGF-BB (rhPDGF-BB) in the Pichia pastoris fermentation broth although during the research of process development and the production manufacture it needs to be stored at low temperature. The concentration of rhPDGF-BB protein in the fed-batch fermentation broth of P. pastoris was stable during 3-week storage at -20 o C, but its bioactivity was reduced by 20%. The addition of a mixture of 50% glycerol with either 1 mM EDTA or 1 mM PMSF into the fermentation broth could fully preserve the bioactivity of rhPDGF-BB until 3 weeks at -20 o C. The addition of 50% glycerol with either 1 mM EDTA or 1 mM PMSF was found no affection on the protein purification process.

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Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity

Cited by 12 publications

References 58 publications

Engineering growth factors for regenerative medicine applications

Engineering growth factors for regenerative medicine applications

Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB

Storage of the Recombinant Protein hPDGF-BB in the Culture of Pichia pastoris

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