title: Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.
abstract: Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Minh Tan
authors.initials: MT
authors.lastname: Nguyen
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Martin
authors.initials: M
authors.lastname: Krupa
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Bon-Kyung
authors.initials: BK
authors.lastname: Koo
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Jung-A
authors.initials: JA
authors.lastname: Song
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Thu Trang Thi
authors.initials: TT
authors.lastname: Vu
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Bich Hang
authors.initials: BH
authors.lastname: Do
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Anh Ngoc
authors.initials: AN
authors.lastname: Nguyen
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Taewook
authors.initials: T
authors.lastname: Seo
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Jiwon
authors.initials: J
authors.lastname: Yoo
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Boram
authors.initials: B
authors.lastname: Jeong
authors.affiliation: Osong Medical Innovation Foundation, New Drug Development Center, Division of Drug Screening and Evaluation, Chungbuk, 363-951, Korea.
authors.forename: Jonghwa
authors.initials: J
authors.lastname: Jin
authors.affiliation: Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, 05505, Korea.
authors.forename: Kyung Jin
authors.initials: KJ
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul 05505, Korea.
authors.forename: Heung-Bum
authors.initials: HB
authors.lastname: Oh
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
authors.forename: Han
authors.initials: H
authors.lastname: Choe
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D058619:Maltose-Binding Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012995:Solubility; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A