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 VEGF(121) 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. VEGF(121) and VEGF(121)/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 VEGF(121) and VEGF(121)/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 VEGF(121) (59.2mg/L) and 27-fold for VEGF(121)/rGel (42.5mg/L), respectively, compared to the conventional flask cultivation method (3.3 and 1.6mg/L for VEGF(121) and VEGF(121)/rGel, respectively). The purified VEGF(121) and VEGF(121)/rGel fusion proteins were biologically active as assessed by phosphorylation of KDR receptors and cytotoxicity against KDR expressing cells.
Kim
Sehoon
S
The Department of Experimental Therapeutics, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Mohamedali
Khalid A
KA
Cheung
Lawrence H
LH
Rosenblum
Michael G
MG
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D019149:Bioreactors; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054789:Ribosome Inactivating Proteins, Type 1; D013552:Swine; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D010940:Plant Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D042461:Vascular Endothelial Growth Factor A; C025674:GEL protein, Gelonium multiflorum