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http://dx.doi.org/10.9713/kcer.2017.55.3.369

Development of Purification Process of Recombinant Human Vascular Endotherial Growth Factor (VEGF) using Fusion Protein  

Sung, Keehyun (Department of Chemical Engineering and Applied Chemistry Chungnam National University)
Kim, In Ho (Department of Chemical Engineering and Applied Chemistry Chungnam National University)
Publication Information
Korean Chemical Engineering Research / v.55, no.3, 2017 , pp. 369-378 More about this Journal
Abstract
Vascular endotherial growth factor (VEGF) is a potent mitogen that stimulates vascular permeability and angiogenesis and has a potential in therapeutic applications. An industrial production method that provides high yield as well as purity is needed. Researches for various factors of mild solubilization with combination of ubiquitin fusion protein to increase solubility were carried out as well as by changing pH and denaturant concentration. Usage of pET28-a bacteral expression vector in BL21 (DE3) host cell was capable of producing approximately 14 g/L VEGF fusion protein in 20L fermentor. A purification process consisting of four chromatography steps including refolding and digestion with UBP1 resulted in mild solublization under the conditions of 2M urea and pH 10.0 due to ubiquitin fusion tag protein that increases in solubility of target protein VEGF. High yield of refolding and dimerization could be obtained between two step Ni-affinity chromatography. Multimeric and misfolded proteins and endotoxin were removed by DEAE anion exchange chromatography. Final monomers were removed from dimers by gel filtration chromatography. Characterization analysis of purified dimeric VEGF was performed using SDS-PAGE and RP-HPLC with a purity of 97%.
Keywords
VEGF; Refolding; Purification process;
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Times Cited By KSCI : 2  (Citation Analysis)
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