Expression And Condition Optimization Of TMP Fusion Protein In Pichia Pastoris

Nowadays,thrombocytopenia is frequently seen in clinic.Howere,there are few effective drugs for this kind of disease.Neotypes of drugs with high effective thrombocytopoiesis are expected.Thrombopoietin(TPO)has been considered to be the principal regulator of megaka-ryopoiesis and platelet production.The effects of TPO on megakaryopoiesis and thrombo-cytopoiesis are mediated by its receptor,c-Mpl,expressed on the surface of megakaryocyte and megakaryoblast cells.Because there was a risk of producing neutralized antibody of TPO for pegylated recombinant human MGDF used in vivo,rhTPO can not be proofed by FDA in U.S.A until now.Recently,a small peptide consisted of 14 amino acids was reported to bind c-Mpl with high affinity and has functions in stimulating proliferation and differentiation of megakaryocytes as same as TPO,while it is heterogenous to TPO,so that it was called TPO-mimetic peptides(TMP).Because of small size,TMP is difficult to be produced by engineering and easily degraded in blood cirulation.Human serum albumin(HSA)is the main protein in the blood,its content is as high as 60%.It can regulate the body's osmotic pressure,balance nutrition and promote wound healing.Due to its relatively large molecular weight(67000)and long half-life in the body,HSA is often considered as an ideal tool to extend biologic half life of small molecular peptides,and is used to construct the fusion protein.In this study,by using four different connecting peptide,we clone the TMP-linker-TMP-HSA to pPink?-HC plasmid through gene recombinate technique.Finally,we successfully build the four different recombinant plasmids which are containing TMP-HSA,and named pPinka-13,14,15,16 respectively.To get the TMP-HSA fusion protein,we transform the recombinant plasmids into four different protease defect type yeast strains respectively,then use the high copy colonies of screening for methanol induced expression,finally,identificate the expression supernatant by SDS-PAGE and Western-blot.The results show that the 16 yeast strains which have been built can specificly express the TMP-HSA secretory fusion protein,and the expression capacity is up to 150 ?g/mL.In the optimum experiment,we use different methanol concentrations and different temperatures to induce the expression of purpose protein,the results show that:the optimum methanol range is 4%-5%and the expression reaches the highest spot at 30? when we express protein in flask with small amount.The results show:expressing the TMP-HSA secretory fusion proteins in P.pastoris is feasible.