Cell proliferation assay of purified hGH in the Nb2-11 cell line.

<p>Dose-response proliferation curve of Nb2-11 cells by exposure to different concentrations of purified hGH and commercial hGH.•, commercial hGH; △, hGH from Trx-hGH; ◊, hGH from Trx-hGH in presence of DTT; □, hGH from MBP-hGH.</p

Expression level and solubility of hGH with seven different tags.

<p>Expression level and solubility of hGH with seven different tags.</p

Schematic representation of domain structure and generation of the MBP-hGH construct.

<p>(A) Vector map of pHMGWA-hGH using the gateway cloning method. Expression of the fusion proteins in <i>E. coli</i> is controlled by the IPTG-inducible T7 promoter, with ampicillin as the selection marker. (B) Schematic structure of the seven fusion proteins His6-, Trx-, GST-, MBP-, NusA-, PDIb′a′-, and PDI-GH (total size). The arrows indicate the TEV protease cleavage site.</p

MALDI-TOF MS analysis of purified hGH from Trx-hGH in <i>E. coli</i> (no DTT during purification).

<p>(A) Tryptic peptide map of hGH (191 aa). (B) MALDI-TOF MS for purified hGH in reducing conditions. (C) MALDI-TOF MS for the purified hGH in non-reducing conditions. MALDI-TOF MS analysis of reduced and non-reduced hGH obtained from MBP-hGH and PDIb′a′-hGH showed similar results (data not shown).</p

Expression analysis of tagged hGH in <i>E. coli</i>.

<p>Expression of full-length hGH was induced by 0.5 mM IPTG at 37°C (A) and 18°C (B). Arrows indicate hGH proteins fused with each tag. M, molecular weight marker; C, total cell protein before IPTG induction as a negative control; I, total cell protein after IPTG induction; P, pellet fraction after cell sonication; S, soluble supernatant after cell sonication.</p

Prokaryotic Soluble Overexpression and Purification of Bioactive Human Growth Hormone by Fusion to Thioredoxin, Maltose Binding Protein, and Protein Disulfide Isomerase

<div><p>Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in <i>Escherichia coli</i> (<i>E. coli</i>) has proven difficult because the <i>E. coli</i>-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′), were tested for soluble overexpression of codon-optimized hGH in <i>E. coli</i>. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb′a′-, or Trx-tagged hGH expressed at 18°C in <i>E. coli</i> using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb′a′-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.</p></div

hGH purification from Trx-hGH expressed in <i>E. coli</i>.

<p>(A) Flowchart of the purification. (B) Trx-hGH was purified from <i>E. coli</i> with a combination of IMAC and gel filtration chromatography. M, molecular weight marker; lane 1, total cell protein before IPTG induction as a negative control; lane 2, total cell protein treated with IPTG; lane 3, soluble fraction after cell sonication; lane 4, Trx-hGH fusion protein purified using IMAC (37.4 kDa); lane 5, Trx tag cleavage with TEV protease: Trx (15.4 kDa) and hGH (22 kDa); lane 6, final purified hGH (22 kDa). Lane 5 shows that the Trx tag was almost completely cleaved. (C) Gel filtration chromatogram of PDIb′a′-hGH after second IMAC. hGH and oligomers were separated by their sizes. (D) Purity of final product hGH was evaluated by silver staining. M, molecular weight marker; hGH: final product in non-reducing conditions.</p

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

<div><p>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. <i>Escherichia coli</i> is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in <i>E</i>. <i>coli</i> has proven difficult because the <i>E</i>. <i>coli</i>-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 <i>E</i>. <i>coli</i>. 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 <i>E</i>. <i>coli</i> 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 <i>E</i>. <i>coli</i>.</p></div

Analysis of hVEGF purification from <i>E</i>. <i>coli</i>.

<p>(A) Schematic overview of hVEGF purification. (B) His6-MBP-hVEGF was purified from <i>E</i>. <i>coli</i> by chromatography. Lane 5 shows that the MBP tag was almost completely cleaved. M, molecular weight marker; lane 1, total cell extract before IPTG induction as negative control; lane 2, total cell extract with IPTG induction; lane 3, soluble fraction after cell sonication; lane 4, His6-MBP-hVEGF fusion protein purified using MBP column (62.9 kDa); lane 5, His6-MBP tag cleavage with TEV protease (28.6 kDa): His6-MBP (43.9 kDa) and hVEGF (19 kDa); lane 6 and 7, purified hVEGF using Heparin column: final hVEGF product under reducing and non-reducing conditions, respectively. The arrows indicate positions of hVEGF as monomer (19 kDa), dimer (38 kDa) and oligomers (≥100 kDa). (C) SDS-PAGE (lanes 1–3) of reduced and non-reduced samplesand Western blot analysis (lanes 4–6) with anti-hVEGF. The arrows indicate the band signals of hVEGF in fusion with MBP tag or in final product. M, molecular weight marker; lane 1 and 4, total cell extract with IPTG induction; lane 2 and 5, final hVEGF product under reducing condition; lane 3 and 6, final hVEGF product under non-reducing condition.</p

Expression analysis of tagged hVEGF in <i>E</i>. <i>coli</i> Origami 2 (DE3) by SDS-PAGE.

<p>Expression of full-length hVEGF was induced by 0.5 mM IPTG at 37°C (A) and 18°C (B). Arrows indicate the target fusion proteins. His6, hexa (poly) histidine; Trx, thioredoxin; GST, glutathione-S-transferase; PDIb'a', b'a' domain of full-length human PDI; MBP, maltose-binding protein; NusA, N-utilization substance protein A; PDI, full-length human PDI; M, molecular weight marker; C, total cell protein before IPTG induction as negative control; I, total cell protein after IPTG induction; P, pellet fraction after cell sonication; S, soluble fraction after cell sonication.</p