Study On The Optmization Of Expression Parameters, The Purification Procedure And The Tumor Cell Multiplication Inhibition Activity Of Recombinant Cucurbita Maxima Trypsin Inhibitor Ⅰ

Cucurbita maxima trypsin inhibitor I (CMTI I) is one kind of the serine protease inhibitors from Cucurbita family which consists of more than 20 members. Because of their low molecular weights, simple structures and clear mechanism, CMTIs are used as tools for the study of the structure-function relationships of active proteins and their mechanism. Though natural CMTIs have clear activities, it is difficult to develope them into drugs to their similarities in molecular weights and structures of different members of the family, which result in the difficulty to get large amount of CMTI I by isolation from Cucurbita seeds. For this reason, the genetic engineering strains of Pichia pastoris GS115 producing recombinant CMTI I ( rCMTI I) was constructed in our lab. In order to carry out deep research and development for rCMTI I, the expression parameters was optmized, the purification procedure was established and the tumor cell multiplication inhibition activity was studied in this paper. The main research contents and results are as foolows.1 Study on the effects of different fermentation parameters on the expression of rCMTI IIn our study the rCMTI I was expressed by reconstructed Pichia pastoris GS115 containing CMTI I gene. Fermentation experiment showed that Mg²⁺ and yeast nitrogen base could increase the expression of CMTI I by 15 %å’Œ 21 % respectively. It was found that the change of pH affects the fermentation obviously and certain pH could promote rCMTI I expression in buffer system. The optimized conditions was determined by orthogonal design as: yeast nitrogen base 1.34%, Tryptone 8%, glucose 8%, 10mg/L Mg²⁺ , pH6.0. The fermentation test carried out with optimized fermentation...