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

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.

Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein

“…Refolding yield of active oligomeric proteins from IBs is even lower (Scrofani et al, 2000; Karumuri et al, 2007; Garrido et al, 2011). Formation of active monomer and its association is a prerequisite for refolding into fully active oligomeric proteins.…”

Expression and Production of Therapeutic Recombinant Human Platelet-Derived Growth Factor-BB in Pleurotus eryngii

“…This is the first report to express hPDGF-BB in P. eryngii by the Agrobacterium-mediated transformation system and demonstrated a possibility to produce pharmaceutically important gene in transgenic mushroom. Previously, expression of hPDGF-BB has been attempted by various researchers using variety of heterologous systems such as bacteria, yeast, insect, and mammalian cells [17][18][19][20][21]32]. The expression level in these systems is a key issue that has led to high production cost.…”

“…The expression level in these systems is a key issue that has led to high production cost. Wang et al [20] reported the yield of 32 mg l −1 from S. cerevisiae whereas Karumuri et al [18] could achieve about 10-12 mg g −1 from E. coli. In the current study using A. tumefaciens, significantly higher yield of recombinant hPDGF-BB (40.4 μg ml −1 of culture processed) was achieved from P. eryngii.…”

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

“…Bioactivity assay-Biological activity of rhPDGF-BB was evaluated by a modified method based on the report of Karumuri et al [4] using the NIH-3T3 fibroblast cell line (American Type Culture Collection) and the Sigma-Aldrich® MTT Cell Proliferation Assay Kit. The cells were cultured in Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 Ham (DMEM/ F12, 1:1 mixture; Himedia) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 1X Antibiotic Antimycotic Solution (100 U ml -1 penicillin, 100 µg ml -1 streptomycin and 0.25 µg ml -1 amphotericin B; Sigma-Aldrich) at 37°C in 5% CO 2 .…”

“…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.…”