Studies On Production Technology Of Recombinant Human Stem Cell Factor

The thesis consists of four sections. A production technology of recombinant human stem cell factor (rhSCF) was mainly studied, including the studies of the culture media of recombinant E.coli, culture conditions being in shaking-flask, batch culture in 5-liter-fermenter, separation and purification of the rhSCF. It was summarized as following.1. Investigation of culture mediaThe culture media of fermentation for the recombinant E.coli DH5a/pBV220 to produce the rhSCF were studied with shaking-flask method. Firstly, the M9 medium was selected from several kinds of basic culture media, and adapted to the growth of bacteria and expression of rhSCF. Secondly, With a single factor experiment, the substances of carbon and nitrogen sources were selected. The results showed that glucose is one of the best carbon sources, and tryptone and yeast extract are good in many nitrogen sources. Thirdly, the recipe of optimized culture medium was obtained by orthogonal test. In addition, the growth of bacteria was found to become fast when magnesium(II) was added in culture broth.2. Optimization of the fermentation conditions with shaking-flaskThe effects of culture temperature, time of inducing, expression times on the growth of E.coli and expression of rhSCF were systematically investigated. Also the process conditions, such as initial pH of culture broth, dissolved oxygen, inoculum age, inoculum volume and so on were optimized. The results showed the optimum conditions : temperature 30℃, initial pH 7.2-7.4, the volume of culture broth in shaking-flask 50mL, approximate rotation speed 180r/min. It was suitable for inducing cultivation at 42℃ when the growth of bacteria was in the initial logarithm growthphase and expressing 3 hours, then shifted to 35℃. Under these culture conditions, the expression and yield of rhSCF in shaking-flasks were 24.6% and 0.276g/L, respectively. Additionally, the fermentative characteristics in shaking-flasks under these optimum conditions were studied and indicated that the insufficient of dissolved oxygen was the mainly reason for the restricting growth of bacteria and expression of rhSCF in the whole fermentation process.3. Batch culture with 5-liter-fermenterBased on the results of shaking-flask cultivation, the batch culture conditions of the rhSCF in 5-liter-fermenter were investigated. The optimum conditions were found as following: the level of dissolved oxygen no less than 50% by adjusted inhalant volume and rotation speed, inoculum volume 10-15%, induced at OD600 2.0. Then the yield of 0.626g/L of rhSCF was obtained, which was enhanced 130% more than the yield of 0.276g/L in shaking-flask. The studies on fermentative characteristics of the rhSCF in the whole batch fermentation process showed that nutrients were sufficient in the end of fermentation, so we can expected that the yield of rhSCF may be further added in fed-batch culture.4. Separation and purification of rhSCFThe inclusion bodies of rhSCF were extracted by different solutions, which were directly injected into high performance hydrophobic interaction chromatography (HPHIC) and high performance ion exchange chromatography (HPIEC). With only one HPHIC step, the purity of rhSCF obtained was 95%, and its mass recovery was 32.6%. Additionally, compared to HPHIC, the purity of rhSCF was lower when the extract was separated by HPIEC.