Cloning, Expression, Fermentation With Shaking-flask And Purification Of Recombinant Human GM-CSF

The thesis includes four parts. 1. Based on PCR and DNA recombination technologies, the recombinant plasmid pMD18-T/hGM-CSF was constructed; 2. The expression of rhGM-CSF in E. coli was also investigated; 3. The optimization of the fermentation conditions with shaking-flask for rhGM-SCF in E. coli was studied; 4. The separation and purification of rhGM-CSF extracted with 8.0 mol.L-1 urea was also investigated with ion exchange chromatography.1. Cloning of human GM-CSFOne pair of primers was designed according to the published sequence of human GM-CSF. Polymerase chain reaction (PCR) and synthesized oligo-nucleotide primers were used to modify the 5' terminal cDNA sequence of human GM-CSF. Then the fragment of hGM-CSF was irtserted into the pMD18-T vector and transformed into E. coli XL10-gold. The recombinant plasmid pMD18-T/rhGM-CSF was tested by PCR, digestion and sequence analysis. The results indicated that the cloned gene fragment of hGM-CSF was the same as that of the human.2. Expression of rhGM-CSFThe modified hGM-CSF was inserted into expression vector pDH by means of DNA recombination in vitro, then the recombinant expression plasmid pDH/rhGM-CSF was constructed and transformed into E. coli DH5 . The expression of rhGM-CSF was induced by elevating culture temperature up to 42 C, and SDS-PAGE of the E. coli lysates showed that the expressed rhGM-CSF constituted about 16% of total cellular proteins in the E. coli lysates3. Optimization of the fermentation conditions with shaking-flaskThe effects of culture media, culture temperature, time of inducing, expression timeson the growth of bacteria and expression of rhGM-CSF were investigated. Also the process conditions, such as initial pH of culture broth, rotational speed, inoculum volume and so on were optimized. The results showed the optimum conditions: temperature 32, initial pH 7.2-7.4, approximate rotation speed 200 r-min-1. It was suitable for inducing cultivation at 42 C for 4-5 hours when the growth of bacteria was in the initial logarithm growth phase. Under these culture conditions, SDS-PAGE indicated that the quantity of rhGM-CSF is 24.3% of total bacterial protein.4. The separation and purification of rhGM-CSFThe separation and purification of rhGM-CSF extracted with 8.0mol-L-1 urea was investigated with weak-anion exchange chromatography (EC). The effects of the composition of the mobile phase on the separation of rhGM-SCF were studied. The results show that with the mobile phase containing a certain ratio of GSH to GSSG and 3.0mol-L-1urea, rhGM-SCF can not only be separated, but also be renatured simultaneously with IEC. As a result, the purity and the mass recovery of rhGM-CSF can be obtained to 95% and 60% by one step with IEC, respectively. Comparing with the standard rhGM-SCF, the same retention time and peak shape can be obtained from IEC. The result indicated that IEC also can be applied to the renaturation with simultaneous purification of rhGM-CSF.