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.

Escherichia coli inner membrane display system for high‐throughput screening of dimeric proteins

“…For analyzing the VH–VL assembly, the MBP‐hVEGF fusion complex was overexpressed in the cytoplasm of E. coli Origami2(DE3; Nguyen et al, ). After affinity chromatography using amylose resin, over 10 mg MBP‐hVEGF was successfully purified from a 500 ml culture (Figure a).…”

“…Human vascular endothelial growth factor (hVEGF) fused with maltose‐binding protein (MBP) was expressed in E. coli as described previously (Nguyen et al, ). Briefly, the pDEST‐HMGWA‐hVEGF plasmid (a kind gift from Prof. Han Choe, University of Ulsan College of Medicine) was transformed into E. coli Origami2(DE3), and a single colony of a transformant was inoculated and cultivated in 5 ml luria‐bertani (LB) supplemented with 50 μg/ml ampicillin for 16 hr.…”

“…Human vascular endothelial growth factor (hVEGF) fused with maltosebinding protein (MBP) was expressed in E. coli as described previously (Nguyen et al, 2016…”

“…In MPDA, monomeric subunit polypeptides are expressed on the periplasmic surface of the E. coli inner membrane, and membrane-tethered polypeptides are spontaneously assembled due to membrane fluidity, facilitating the display of complex multimeric proteins for highthroughput screening. We showed that membrane-anchored poly- This study pPelB-AglycoT(H)-Fc-FLAG Trastuzumab H chain gene in pPelBFLAG (Jung et al, 2013) pPelB-AglycoT(H)-Fc1004-FLAG Trastuzumab Fc1004 mutant H chain gene in pPelBFLAG (Jung et al, 2013) pPelB-AglycoT(H)-MG48-FLAG Trastuzumab MG48 mutant H chain gene in pPelBFLAG (Jo et al, 2018) pAK200 Cm r , lac promoter, tetA gene, C-terminal gpIII gene fusion (Krebber et al, 1997) pDEST-HMGWA-hVEGF Amp r , T7 promoter, N-terminal 6x polyhistidine tag, MBP-hVEGF gene (Nguyen et al, 2016) Note SfiI endonuclease, ligated into pMopac12-NlpA-FLAG and pAK200 (Krebber et al, 1997), and digested with the same restriction enzyme.…”

Guidelines to reach high-quality purified recombinant proteins

“…On the other hand, SDS-PAGE may detect protein degradation (smeared bands of lower MW than the target protein), glycosylation (using specific staining) and, in some cases, oligomerization. For example, recently, SDS-PAGE gel stained with Coomassie brilliant blue R-250 revealed monomeric (19 kDa), dimeric (38 kDa) and oligomeric (> 100 kDa) forms of a human growth factor (VEGF) recombinantly produced from E. coli, by analysis of a protein sample prepared under non-reducing conditions (Nguyen et al 2016). SDS-PAGE can also be used to purify a protein (through band excision) from a partly purified protein sample for analysis of its primary structure by Edman degradation (Nterminal sequencing) (Oliveira et al 2008) Fig.…”