Study On The Isolation And Purification Procedure And The Activity Of Recombinant Cucurbita Maxima Trypsin Inhibitor â… 

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 because of their similarities in molecular weights and structures of different members of the family, which results in the difficulty to get large amount of CMTI-I by isolation from Cucurbita maxima 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 isolation and purification procedure was established and the tumor cell multiplication inhibition activity was studied in this paper. The main research contents and results are as follows.1 Preparation of rCMTI-I affinity chromatography mediumIn order to establish a cheap isolation and purification procedure for rCMTI-I by affinity chromatography, the preparation of the affinity medium was studied. First, the affinity medium was prepared by activating microcry stalline cellulose for column chromatography use with epichlorohydrin and coupling trypsin as the lingand to the matrix. Meanwhile activating conditions and the use of trypsin for the affinity medium were studied. The best condition for activation is using a mixture solvent of 2.4mol/L NaOH solution and DMSO(volume rate is 1: 1), at 30℃, activating for 3 hours, using 20ml of epichlorohydrin for microcry stalline cellulose of 1g. The epoxy density of activated microcry stalline cellulose under the best condition was (302.4908±10)μmol/g.2 Establishment of rCMTI-I purification procedure Rcombiment CMTI-I was isolated directly from the culture medium by using the prepared affinity medium. The resulting rCMTI-I sample was desalted by Sephadex G-10 which could remove the low molecular weight materials of rCMTI-I and salt at the same time and further purified by Sephadex G-25 chromatography. Pure rCMTI-I was obtained. Affinity chromatography was more effective compared with ion exchange chromatography.3 Study on the tumor cell multiplication inhibition activity of rCMTI-IThe in vitro study of tumor cell multiplication inhibition activity of rCMTI-I showed that rCMTI-I could significantly inhibit SKOV3 cell line and SE-2 cell line, and the action had a dose-dependent effect with inhibition rates of 69.3% and 86.5% for the two cell lines respectively at the concentration of 3.82×10^-2μmol/ml. The results suggest that rCMTI-I may have anti-tumor activity.4 Study on the MMPs inhibition activity of rCMTI-IThe inhibition activity of rCMTI-I on MMPs was tested with TNBSA assay, which showed a dose-dependent effect. The results suggest that rCMTI-I may have anti-tumor metastasis effect.The main achievements obtained in the study are as follows:a. The isolation and purification procedure of rCMTI-I from the supernatant of cultured medium was established on the basis of affinity medium preparation and investigation. The procedure is suitable for large scale production.b. The tumor cell multiplication inhibition activity on tumor cells of rCMTI-I was found, which gave us a new thought to study the anti-tumor activity of rCMTI-I.c. The activity of rCMTI-I on MMPs was tested with TNBSA assay, which showed an inhibition activity.The study has laid the foundation for the development of rCMTI-I as a new drug.