The Research Of Pilot-scale Ferment TMP Fusion Protein In Pichia Pastoris

Nowadays there have been thousands of people with thrombocytopenia caused by idiopathic thrombocytopenic purpura(ITP),secondary thrombocytopenic purpura and chemotherapy-induced thrombocytopenia in our country.Traditional therapeutic method such as platelet transfusion would cause side effects like immunoreaction,secondary infection which damage to health of patients,and increase financial burden to families.Thrombopoietin(TPO)is a main regulatory factor in the production and differentiation of plateletsand megakaryocytes.Thrombopoietin was cloned in 1994,then scientists gained much interests in its clinical applications.But because of neutralizing antibody produced in endogenous TPO cross reaction which cause reduce of platelet continuous,it has been stopped in clinical test in America.TMP is a TPO analogue with 14 amino-acids which have nothing nucleotide sequence homology to TPO.It is reported TMP can competitive bind to c-mpl and promote the production of platelet.TMP dimer have the same function as TPO,and also it would not induce neutralizing antibody.But short shelf life prevent its use in promoting platelet production.Fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics which conjugate the gene of polypeptides or protein with short shelf life to specific carrier protein gene.Fusion protein expressed by fusion gene still with functional properties also long shelf life.In present work,HSA-TMP fusion protein expressed in Pichia pastoris had been successfully constructed,and then a preliminary exploration for ferment condition of fusion protein expression in pilot-scale had been done.After the study of pichia growth curve,the effect of BSM in yeast grow,and different induction condition in the biomass of target protein.The optimal pilot-scale fermentation conditions:cultured the Pichia Pastoris at 29℃,methanol induced at 27℃,pH=5,adjusting parameters to ensure DO ≥ 20%.In first step pichia was cultured approximate 36 hours until glycerin exhausted,then into second step,methanol induced phase.After 96 hours fermentat:ion,The biomass of Pichia up to 500 g/L,and at this time the fusion protein HSA-TMP yield 1 g/L and the better bioactivity.Taken together,we obtain preliminary conditions for pilot-scale fermentation fusion protein HSA-TMP by Pichia expression system.we make a meaningful attempt for the thrombocytopenia in research and therapy.