Cloning And Expression Of Human Vascular Endothelial Growth Factor Recptor-2(KDR) Gene In Streptomyces Lividans TK24 And E.coli BL21 And Study Of The Screening Model For KDR Inhibitors

Angiogenesis is defined as the development of new blood vessels from preexisting vessels,and it is a crucial process not only in normal physiology but also in pathological process such as embryonic development,wound healing,the normal menstrual cycle and tumor metastasis and tumor-associated blood vessels.Especially angiogenesis plays an important role in solid tumor growth and tumor metastasis. Vascular endothelial growth factor(VEGF) acts as a highly specific mitogen directly involving in the process of angiogenesis,which induces endothelial cells division and proliferation and also increases vascular permeability.VEGF performs its function through bindig to special receptors.Three high-affinity cognate endothelial receptors for VEGF have been identified:VEGFR-1/Flt-1,VEGFR-2/Flk-1/KDR,and VEGFR-3/Flt-4.These receptors belong to the subfamily of classâ…¢receptor tyrosine kinases(RTKs).Studies have indicated that KDR is the major signal transducer for the differentiation and proliferation of endothelial cells.VEGF binding to KDR results in dimerization of receptor monomers,transphosphorylation by dimerized receptors and docking of signaling proteins to receptor phosphotyrosines.An increase in the intrinsic catalytic activity and creation of binding sites on the RTKs to recruit cytoplasmic signaling proteins are primary features of RTKs activation,resulting in endothelial cells-associated reaction.As angiogenesis is of crucial importance for tumor growth, effectively inhibiting KDR signaling is considered as an attractive target for drugs design and screening novel anticancer agents against tumor angiogenesis.A full-length 5821bp KDR cDNA includes 4068bp-coding region encoding 1356 amino acids,whose encoding gene locates in 4q12.KDR is characterised by seven extracellular immunoglobulin(Ig)-like domains followed by a membrane-spanning region and a conserved intracellular tyrosine kinase domain.Extracellular region is composed of 766 amino acids,2-3 Ig-like domain are main binding sites for VEGF. Intracellular tyrosine kinase domain has 565 amino acids,consisting of a kinase domain split by an about 68-amino-acid kinase insert,and a carboxyl terminus. With RNA extracted from human umbilical vein endothelial cell as the template, the KDR cDNA encoding 1-3 extracellular immunoglobulin(Ig)-like domains (KDR-ED) and the catalytic core in intracellular tyrosine kinase domain(KDR-CD) were amplified with RT-PCR.The sequence of amplified KDR-ED and KDR-CD was completely identical with the published data(GenBank Accession No.AF063658).Using the shuttle plasmid(Streptomyces-E.coli) pSGLgpp as expression vector, KDR-ED and KDR-CD were cloned and expressed in S.lividans TK24 separately. After the gene KDR-ED and KDR-CD were cloned at the downstream of gpp signal peptide in the plasmid pSGLgpp separately,the recombinant plasmids were transformed into S.lividans TK24,and the strains were named as S.lividans[pSGLgpp/KDR-ED]and S.lividans[pSGLgpp/KDR-CD]respectively. SDS-PAGE showed that a special protein band with MW 44kD appeared on the gel using the expressed protein of S.lividans[pSGLgpp/KDR-CD].With an anti-phosphotyrosine antibody,the result of Western blotting of the recombiant KDR-CD identified a protein band of about 44 kD on the membrane.Such results demonstrated the recombiant KDR-CD expressed by S.lividans has immunonogical activity and the protein was already phosphated in Streptomyces. S.lividans[pSGLgpp/KDR-ED]could express recombinant KDR-ED with about MW 37kD.At the meantime,with the plasmid as pET-30a as vector,the gene of KDR-ED and KDR-CD were cloned and expressed in E.coliBL21(DE3) seperately.With SDS-PAGE, the apparent molecular weight of expressed KDR-CD was about 41kD existing in inclusion body form mostly.With an anti-phosphotyrosine antibody,the result of western blotting showed that the observed protein band of about 41kD in whole pellet, the soluble fraction and the inclusion body,respectively.The results confirmed that the recombinant KDR-CD protein from E.coli has immunological activity and the protein was phosphated in E.coliBL21(DE3).With SDS-PAGE,the KDR-ED was not expressed in E.coli. The recombinant KDR-CD expressed in E.coli and S.lividans were purified, respectively.The KDR-CD was purified from the supernant of S.lividans[pSGLgpp/KDR-ED]by the following steps:70%ammonium sulfate precipitation,cation exchange resin SP-sepharose Fast Flow and an anion exchange resin Q-sephorose Fast Flow.After the recombinant KDR-CD protein from E.coli[pET-3Oa/KDR-CD]was denatured in urea.Through renatured treatment and Ni-NTA affinity chromatograph,the purified KDR-CD protein was obtained.The result of SDS-PAGE showed that the recombinant KDR-CD,which was obtained from two kinds of prokaryotic hosts using different purification methods,was pure.HPLC analysis showed that the purify reached 95%,respectively.Using ELISA method,tyrosine kinase activity of purified KDR-CD from two kinds of prokaryotic hosts was assayed,respectively.In these assays,poly(E4Y) was used as reacting substrate.The optimal condition was tested including the quantity of KDR-CD,the condition of substrate and ATP and the condition of Mg²⁺ and Mn²⁺. Results showed that KDR-CD from two kinds of prokaryotic hosts had tyrosine kinase avtivity.With the purified KDR-CD as target,the screening model for tyrosine kinase inhibitors was constructed.More than 600 microbiological metabolites were screened. Among them,13 metabolites were found with the inhibiting activity and the positive rate was about 2.17%.As showing above,KDR-CD was expressed secretly in Streptomyces,which has not reported before.With the recombinant KDR-CD from Streptomyces as target,a novel drug-screening model for inhibitors of tyrosine kinase has been established, which is more effective practice and creative.It will lay on good foundation for seeking novel drugs for anti-tumor angiogenisis.