Expression of Human Vascular Endothelial Growth Factor (VEGF_(165)) in Pichia pastoris and Its Biological Activity

Objective To express human vascular endothelial growth factor (hVEGF-{165}) cDNA in %Pichia pastroris, %purify the expressed product and detect the biological activity of it. Methods By inserting hVEGF-{165} cDNA coding 165 amino acid residues into %Pichia pastoris% expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/hVEGF-{165} was constructed and transformed to yeast host strain KM71, then multiple_copy insert transformants were screened out and cultured in flasks, and hVEGF-{165} was expressed under the induction of 1% methanol. Results SDS_PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by Heparin_Sepharose CL6B affinity chromatography, the purity of expressed product reached above 90%. Biological assays proved that the expressed product could stimulate the proliferation of HUVEC. Conclusion hVEGF-{165} was successfully expressed. The study opened up a wide prospect for the application of VEGF-{165} in the prevention and treatment of ischemic heart disease and other tissue ischemic diseases such as secondary arterial occlusion in limbs.

Expression of Human Vascular Endothelial Growth Factor Receptor Flt-1 Extracellular Domain 1-3 Loop cDNA in Pichia pastoris, Purification of the Expressed Product and Detection of Its Biological Activity

Objective To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. pastroris, and to purify the expressed product and detect its biological activity. Methods By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His + Mut s phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol. Results SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF 165 and inhibit the proliferation of HUVEC stimulated by hVEGF 165. Conclusion Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line

Background The genome of the severe acute respiratory syndrome-associated coronavirus ( SARS-CoV) includes sequences encoding the putative protein X4 ( ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection. Methods The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles. Results We expressed and purified soluble recombinant protein X4 from E. coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the tung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 313 cells as determined by cell counting and MTT assays. Conclusion The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.