title: Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink.
abstract: The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 μg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.
authors.affiliation: Baqiyatallah University of Medical Sciences Applied Biotechnology Research Center Tehran Iran.
authors.forename: H
authors.initials: H
authors.lastname: Babavalian
authors.affiliation: Baqiyatallah University of Medical Sciences Applied Biotechnology Research Center Tehran Iran amlatifi290@gmail.com.
authors.forename: A M
authors.initials: AM
authors.lastname: Latifi
authors.affiliation: Pasteur Institute of Iran National Cell Bank of Iran Tehran Iran.
authors.forename: M A
authors.initials: MA
authors.lastname: Shokrgozar
authors.affiliation: Pasteur Institute of Iran National Cell Bank of Iran Tehran Iran.
authors.forename: S
authors.initials: S
authors.lastname: Bonakdar
authors.affiliation: Baqiyatallah University of Medical Sciences Applied Biotechnology Research Center Tehran Iran.
authors.forename: H
authors.initials: H
authors.lastname: Tebyanian
authors.affiliation: Baqiyatallah University of Medical Sciences Applied Biotechnology Research Center Tehran Iran.
authors.forename: F
authors.initials: F
authors.lastname: Shakeri
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000077214:Becaplermin; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020574:Proto-Oncogene Proteins c-sis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification; D012015:Reference Standards; D015203:Reproducibility of Results; D014170:Transformation, Genetic
chemical_list: D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin