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Study on the Purification and Biological Functions of Human Vascular Endothelial Growth Factor and Its Receptor Expressed from Eschicheria Coli

Author: LiuLiCheng
Tutor: ChenQiang
School: Lanzhou University
Course: Physiology
Keywords: hVEGF sFlt-1 Sarkosyl GST pGEX-4T-1 HPLC Bingding Assay CAM
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 120
Quote: 0
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Abstract


The growth and metastases of solid tumors are dependent on the angiogenesis,a procedure of new blood vessel growth in solid tumors and a procedure mediated by numerous inhibitors and activators.Among the activators,VEGF is one of the most potent angiogenic factors.and also is the most widely researched angiogenic factor.The VEGF165 and the Ig-like 1-3 domains of sFlt-1 gene fragments were amplified by RT-PCR from Human placenta cDNA.The PCR fragments encoding VEGF165 and sFlt-1 were cloned into prokaryotic cloning vector pMD19-T and then transformed into E.coli DH5α.The recombinant cloning plasmids pMD19-T/VEGF165、pMD19-T/sFlt-1 and pGEX-4T-1 prokaryotic expression vector wre digested with upstream EcoRI and downstream NotI,and then the products VEGF165 and sFlt-1 were cloned into pGEX-4T-1 using Roche 5 minutes rapid reaction kit.The fidelity of recombinant recombinant expression plasmids pGEX-4T-1/VEGF165 and pGEX-4T-1/sFlt-1 were finally confirmed by Double digestion method and DNA sequencing.The recombinant expression plasmids were then transformed to E.coli strain BL21（DE3）pLysS.The recombinant E.coli was induced by the addition of IPTG to make a final concentration of 1mM and the culture was further incubated at 37℃for 2 or 3 hours.The recombinant E.coli expressed GST-VEGF165 and GST-sFlt-1 at the level up to 39.5%and 53.9%of the total proteins.For inclusion body lysis,afer 100μg/ml lysozyme cleavage E.coli cells,1.5%Sarkosyl was added to the E.coli lysates.After sonication,the lysate was clarified by centrifugation at 13000rpm/min for 10 min.The supernant was transferred to a new tube and treated with TritonX-100 at a final concentration of 3%.After affinity purification by glutathione Sepharose 4B,the purified GST-VEGF165 and GST-sFlt-1 may be achieved 90.6%and 100%.The overall yield was approximately 8-12mg/ml.The HPLC assay showed the purified GST-VEGF165 exists mainly in the form of monomers and dimmers.The Western blot assay showed purified GST-VEGF165 and GST-sFlt-1 have the ability to combine their specific antibodies.The Chick chorioallantoic membrane test showed the purified GST-VEGF165 has the ability to induce angiogenesis.The binding assay showed the GST-VEGF165 binds to VEGF receptor in a dose-dependent manner. The traditional protein purification method contais inclusion bodies cleaning、inclusion bodies solubilization、proteins purification and refolding.Our current work provides a novel procedure for solubilization and purification of GST-fusion proteins:VEGF165 and sFlt-1 in the pGEX-4T-1 fusion system,and the fusion proteins were solubilized and purified with the alkyl anionic detergent,Sarkosyl.Using this method,the traditional three-step method was improved into a two-step method containing inclusion bodies solubilization and protein purification.Our work,for the firt time,provides a simple,effective and time-saving procedure for solubilization and purification of GST-fusion proteins.

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