Optimization of a Purification Method for the Recombinant Platelet-Derived Growth Factor rhPDGF-BB Expressed in the Methylotrophic Yeast Pichia Pastoris

IF 1 4区生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Applied Biochemistry and Microbiology Pub Date : 2023-08-12 DOI:10.1134/S0003683823040087

A.-A. V. Misterova, V. A. Chicherin, A. S. Gerasimov

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Abstract

Recombinant human platelet-derived growth factor rhPDGF-BB is one of the major cytokines that has been approved for medical use. Medical drug becaplermin, which contains rhPDGF-BB, has been approved for neuropathic ulcer and severe skin burns treatment, as well as in periodontal surgery (in combination with osteoconductive matrices). In this article, we sought to optimize the purification process to obtain high purity rhPDGF-BB using methylotrophic yeast Pichia pastoris, the production host for rhPDGF-BB. A faster and simpler chromatography purification method has been suggested that allows one to obtain rhPDGF-BB with a purity of more than 98% as determined by SDS-PAGE containing host cell proteins (HCPs) at 33 ± 4 ng/mg, as measured by ELISA. The effective proliferative dose of rhPDGF-BB measured by the WST-1 proliferative assay on 3T3 mouse fibroblast cell culture is 5.02 ± 2.64 ng/mL, which is comparable to commercially available analogues. The optimized method can be attractive for production scale use.

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甲基营养酵母毕赤酵母中表达重组血小板衍生生长因子rhPDGF-BB的纯化方法优化

重组人血小板衍生生长因子rhPDGF-BB是已被批准用于医疗用途的主要细胞因子之一。含有rhPDGF-BB的医用药物becaplermin已被批准用于神经性溃疡和严重皮肤烧伤治疗，以及牙周手术(与骨传导基质联合使用)。在本文中，我们试图优化纯化工艺，以甲基营养酵母毕赤酵母(rhPDGF-BB的生产宿主)为原料获得高纯度的rhPDGF-BB。提出了一种更快速、更简单的色谱纯化方法，可以获得纯度大于98%的rhPDGF-BB，通过SDS-PAGE测定，含有宿主细胞蛋白(HCPs)，浓度为33±4 ng/mg, ELISA测定。WST-1增殖试验测定的rhPDGF-BB对3T3小鼠成纤维细胞培养的有效增殖剂量为5.02±2.64 ng/mL，与市售类似物相当。优化后的方法具有生产规模应用的吸引力。

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来源期刊

Applied Biochemistry and Microbiology 生物-生物工程与应用微生物

CiteScore

1.70

自引率

12.50%

发文量

审稿时长

6-12 weeks

期刊介绍： Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.