Overexpression of biologically active VEGF121 fusion proteins in Escherichia coli

doi:10.1016/j.jbiotec.2006.11.027

Journal of Biotechnology

Volume 128, Issue 3, 20 February 2007, Pages 638-647

https://doi.org/10.1016/j.jbiotec.2006.11.027 Get rights and content

Abstract

Vascular endothelial growth factor-A (VEGF) exists as five different isoforms, which exert their growth stimulatory effects through interaction with the FLK and KDR receptors. The VEGF₁₂₁ isoform has been employed as a highly selective carrier of therapeutic agents to target tumor endothelial cells resulting in inhibition of tumor growth and metastasis. VEGF₁₂₁ and VEGF₁₂₁/rGel fusion toxin containing hexa-histidine tags were expressed in Escherichia coli AD494 (DE3) pLysS. Media containing glycerol as a primary carbon source increased the specific expression levels of soluble VEGF₁₂₁ and VEGF₁₂₁/rGel (mg/L/OD10) by more than two-fold over LB media when grown in a batchtype cultivation in a bioreactor. High cell densities over OD 40 were achieved using a fed-batch method and employing feeding medium containing glycerol and yeast extract. The overall production of the target proteins was improved 18-fold for VEGF₁₂₁ (59.2 mg/L) and 27-fold for VEGF₁₂₁/rGel (42.5 mg/L), respectively, compared to the conventional flask cultivation method (3.3 and 1.6 mg/L for VEGF₁₂₁ and VEGF₁₂₁/rGel, respectively). The purified VEGF₁₂₁ and VEGF₁₂₁/rGel fusion proteins were biologically active as assessed by phosphorylation of KDR receptors and cytotoxicity against KDR expressing cells.

Introduction

The cytokine vascular endothelial growth factor (VEGF)-A has been known to play a central role in angiogenesis and neovascularization of various solid tumors (Veenendaal et al., 2002, Ran et al., 2005, Roy et al., 2006). VEGF receptors Flt-1/FLT-1 (VEGFR1) and Flk-1/KDR (VEGFR2), two members of the VEGF receptor family, are excellent targets to develop therapeutic agents to inhibit tumor growth and metastasis because they are present at high levels in endothelium of tumor vessels while at low levels on normal or mature vascular endothelium (Plate et al., 1992). VEGF-A has several isoforms, ranging from 121 to 206 amino acids. The smallest isoform, VEGF₁₂₁, contains the full biological activity of the larger variants and binds only to Flt-1/FLT-1 and Flk-1/KDR. In addition, unlike the other VEGF-A isoforms, VEGF₁₂₁ does not bind heparin or require heparin for binding to its receptors. These features make VEGF₁₂₁ a particularly attractive carrier for selectively delivering biological toxins to tumor endothelium (Veenendaal et al., 2002, Ran et al., 2005).

Recently, our laboratory reported the construction of a fusion protein composed of VEGF₁₂₁ and the plant toxin recombinant gelonin (rGel) (Veenendaal et al., 2002). Recombinant rGel is a single-chain N-glycosidase similar in its action to ricin A chain (Stirpe et al., 1980). Gelonin cleaves a conserved adenine in 28S ribosome subunit resulting in irreversible inhibition of protein synthesis (Provoda et al., 2003, Sandvig and Deurs, 2005). We have shown that VEGF₁₂₁/rGel strongly inhibits the growth of human bladder cancer (Mohamedali et al., 2005) and metastatic breast tumor cells (Ran et al., 2005) in xenograft models by directing cytotoxic effects against tumor neovasculature. The vascular targeting property of VEGF₁₂₁/rGel may also be useful to treat non-malignant neovascular diseases such as ocular neovascularization, which occurs in diabetic retinopathy or macular degeneration (Akiyama et al., 2005).

Escherichia coli cells have been widely utilized for production of the recombinant fusion proteins (Jeong and Lee, 2001, Jurado et al., 2002, Yuan and Hua, 2002, Richard et al., 2004, Santala and Lamminmäki, 2004, Woestenenk et al., 2004, Sandee et al., 2005). A few reports now are available that have expressed soluble VEGF fusion proteins in a bacterial expression system. For instances, Arora et al. (1999) and Hotz et al. (2002) reported that VEGF₁₆₅ proteins fused with diphtheria toxin (DT, 390 aa) and GST were expressed as soluble proteins in E. coli SG12036. Our laboratory also reported that soluble VEGF₁₂₁/rGel fusion protein could be successfully expressed in E. coli AD494 (DE3) pLysS (Veenendaal et al., 2002, Ran et al., 2005). In both cases, the total expression levels of the fusion proteins were limited by the induction conditions at low cell density (OD ≤ 4.0 at 600 nm) using LB medium in flask level cultivation.

In addition to our laboratory, several groups have also expressed VEGF or its fusion proteins with a His-tag (Siemeister et al., 1996, Scrofani et al., 2000), thioredoxin (Backer and Backer, 2001) and glutathione-S-transferase (GST) (Morera et al., 2006) using E. coli strains such as BL21, BL21 (DE3), and BL21 (DE3) pLysS. Unlike VEGF₁₂₁/rGel, these fusion proteins are mostly expressed as inclusion bodies rather than as soluble proteins. In general, refolding yields are low and the refolded proteins may not always be biologically active.

In this study, we sought to achieve high-level production of VEGF₁₂₁/rGel to meet demands for clinical trials and a large-scale production. The medium composition and cultivation methods were optimized for high expression of soluble VEGF₁₂₁ and VEGF₁₂₁/rGel in E. coli AD494 (DE3) pLysS. The cultivation methods will be applied for producing other VEGF-related fusion proteins using bioreactors.

Section snippets

Cloning of VEGF₁₂₁ and VEGF₁₂₁/rGel in E. coli expression vector pET-32a (+)

The following E. coli host strain was used in this study: AD494 (DE3) pLysS, [Δara⁻, leu7967, ΔlacX74, ΔphoA, Pvu II, phoR, ΔmalF3, F’ [lac⁺, (lacI^q), pro], trxB::kan(DE3)], pLysS (Amp^R, Kan^R, Cam^R). The cloning method for VEGF₁₂₁/rGel has been previously described (Veenendaal et al., 2002). The pET-32a (+) plasmid encoding VEGF₁₂₁/rGel was used as the template for construction of the VEGF₁₂₁ plasmid so that VEGF₁₂₁ was expressed as a thioredoxin fusion protein with a six-histidine (His) tag

Batch cultures of E. coli to produce VEGF₁₂₁ and VEGF₁₂₁/rGel

For 4-L flask cultivation with LB medium, the final cell density of the cultures reached OD 3.5 and the specific expression level of His-tagged VEGF₁₂₁/rGel was found to be 4.5 mg/L/OD₁₀. In 3 or 7 L bioreactors with LB medium, the cell density and the specific expression level of the target proteins were found to be identical for flask cultivation (data not shown). There were no differences in cell growth and protein expression both in flask and bioreactor systems, which might be valid for

Conclusion

The target proteins, His-tagged VEGF₁₂₁ and VEGF₁₂₁/rGel, were successfully expressed in soluble forms by E. coli AD494 (DE3) pLysS.

Using bioreactors, high expression of the target proteins was achieved using GY medium, which we found to be superior to LB medium in flask cultivation. The carbon source was optimized in terms of total soluble protein expressed. Glycerol was selected as the appropriate primary carbon source in this study because its use resulted in optimal expression of the target

Acknowledgment

Research conducted, in part, by the Clayton Foundation for Research.

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Overexpression of biologically active VEGF121 fusion proteins in Escherichia coli

Abstract

Introduction

Section snippets

Cloning of VEGF121 and VEGF121/rGel in E. coli expression vector pET-32a (+)

Batch cultures of E. coli to produce VEGF121 and VEGF121/rGel

Conclusion

Acknowledgment

Protein Express. Purif.

J. Biotechnol.

Protein Express. Purif.

J. Gastrointest. Surg.

Protein Express. Purif.

J. Mol. Biol.

Neoplasia

J. Biol. Chem.

Neoplasia

FEBS Lett.

J. Immunol. Meth.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Protein Express. Purif.

Overexpression of biologically active VEGF₁₂₁ fusion proteins in Escherichia coli

Cloning of VEGF₁₂₁ and VEGF₁₂₁/rGel in E. coli expression vector pET-32a (+)

Batch cultures of E. coli to produce VEGF₁₂₁ and VEGF₁₂₁/rGel