Prevention of aggregate formation through mechanism analysis in refolding of recombinant pertactin from Escherichia coli |
| |
| Affiliation: | 1. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China;2. University of Chinese Academy of Sciences, Beijing 100049, PR China;3. Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing 211800, PR China;1. State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, National Engineering Research Center of South China Sea Marine Biotechnology, Department of Biochemistry, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People''s Republic of China;2. Beijing University of Chinese Medicine, 11 Dong Shan Huan Road, Beijing 100029, People''s Republic of China;1. Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 61500, Mexico;2. Departamento de Alimentos, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antoquia, AA 1226, Medellín 050010, Colombia;3. Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, 14 Sur 6301, CU, Puebla, Pue 72570, Mexico;4. Laboratorios Silanes S.A. de C.V., Amores 1304, Col. del Valle, Cd. de México, Mexico;1. Laboratory of Sustainable Biotechnology (LIBioS), National University of Quilmes, Roque Sáenz Peña 352, Bernal B1876BXD, Argentina;2. National Scientific and Technical Research Council (CONICET), Godoy Cruz 2290, CABA C1425FQB, Argentina;1. College of Food Science and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China;2. College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China;1. ENSTA ParisTech, 828, Boulevard des Maréchaux, 91120 Palaiseau, France;2. Mathematical Institute, Andrew Wiles Building, University of Oxford, OX2 6GG, UK |
| |
| Abstract: | Production of natural pertactin for pharmaceutical use is limited by its low abundance. In this study, recombinant pertactin was highly expressed in the form of inclusion bodies in E. coli. However, up to 75% of the soluble turned out as aggregates when refolding by pulse-fed batch dilution. The conceivable route for aggregate formation was proposed as that the C-terminus of partially folded intermediate with a strong hydrophobic core would intertwine with that region of newly added denatured protein, resulting in aggregation between proteins with different folding states. The key factor for prevention of aggregate formation was to improve the synchronization of refolding. For this purpose, flash-batch dilution was conducted at a scale of 5 L and achieved a monomeric refolding yield of above 70%. Aggregates formed were efficiently removed along with impurities by one-step chromatography of Ni-resin. The purity of monomeric pertactin was >98%. An overall yield was 320 mg per liter fermentation liquor with a total recovery of about 59%. The purified protein was characterized by MALDI-TOF, circular dichroism, fluorescence, HPLC and DSC, and showed similar physiochemical properties compared to its natural counterpart. Animal study showed similar immunological responses and antibodies elicited demonstrated a comparable reactivity. |
| |
| Keywords: | Recombinant pertactin Refolding Aggregates Dilution |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
