Overexpression of biologically active VEGF₁₂₁ fusion proteins in Escherichia coli

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.