Sudies On Thrombopoietin Site-directed Mutagenesis And Its Expression In Pichia Pastroris

In this study, thrombopoietin was successfully expressed in the Pichia Pastoris expression system. Furthermore, investigation for TPO expression purification, small scale fermentation and activities has been carried out. The work about this subject had not been reported in literature to date. According to cDNA sequence encoding for TPO mature peptide and MF a signal sequence in the integration vector of Pichia Pastoris, two pairs of primers were designed for amplification of cDNA encoding for TPO mature peptide from plasmid pUH-TPO containing the cDNA encoding human full length TPO integration plasmids pPICZ a AL-TPO for expression integration vector pPICZ a A. Identification by digestion with endonuclease showed that the genes of interest were correctly cloned into the mutiple sites of vectors and DNA sequencing indicated that the DNA sequence was consistent with that reported in literature except for the first nucleotide of codon encoding for 301 proline and that genes of interest were correctly insert in ORF of vectors. Site-specific mutation of the first nucleotide of codon encoding for TPO 301 amino acid was carried out by using site-directed mutagenesis technique. Then, we constructed an integration plasmid pPICZ a AL-TPOm by using Pichia Pastoris vector pPICZ a A and transformed it into Pichia Pastoris GS11S strain. 3 After selection of transformants with Mut phenotype and two-layer-filter screening system, positive transformants secreting TPO mature peptide were cultured and induced by methanol. SDS-PAGE and Western-blotting analysis for culture conditional medium indicated that molecular weight of TPO mature peptide expressed by Pichia Pastoris is about 66KD. Observation for different inducing condition showed that expression level reached the highest at about dayS after 0.5% concentration of methanol inducing expression and optimal conditions were about 1% concentration of methanol, 28-290, pH6.0, flask shaked at more than 200rpm. Secreted expression level of TPO mature peptide was account for approximately 60% of total secreted protein and was about 0.9 ing/L. Primary activity analysis of colony forming unit of megakaryocyte(CFU-Meg) in murine bone marrow suggested that the expressed product can stimulate the formation of CFU-Meg.