| Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) is a kind of hemopoietic growth factor that is composed of 127 amino acids. Its main biological functions are to stimulate the proliferation and differentiation of hemopoietic precursors and enhance the mature effector cells (such as neutrophilic granulocyte, acidophilic cell and mono/macrophage). In clinical applications, hGM-CSF was used as an assistant therapeutic medicine in bone marrow transplantation, cancer, AIDS and leucocytopenia and so on. However, there are still some problems for its clinical applications, for example, low specific activity, large dosage for therapy and short cyclic half-life. Clinical experiments have demonstrated that the hGM-CSF expressed by yeast system was low toxicity and small side effects comparing that expressed by E.Coli. For the natural hGM-CSF, its molecular weight is different between 14.5 and 32 KD due to its different glycosylation levels, and the recent researches showed that the activity of no glycosylation hGM-CSF was higher. In our work we firstly used the primer introducing point mutation method and fished out the gene, then mutated two O-glycosylation sites that the 9-Ser and 10-Thr were changed by 9-Ala and 10-Ala. Restriction enzyme analysis and DNA sequencing was carried out, the DNA sequences proved to be the same as designed. Natural hGM-CSF and mutant hGM-CSF gene fragment have been inserted into MCS of the plasmid pPIC9K differently. We transferred the recombinant plasmids into E.coli strain Top10F' to amplify plsmids. Then introduced the linearized plasmids into Pichia Pastoris. Only the recombinant yeast strain could survive on the His- plates. And the gene copy number is coincidence with the ability of anti-G418 and expression level in Pichia Pastoris. So selected the higher anti-G418 strain which also was the higher gene copy numbers and had higher expression level. Furthermore some optimized expression conditions had been explored. In our experiments the control was the wild type hGM-CSF gene expressed in Pichia Pastoris. In recent years Pichia Pastoris was a major expression system, it have many advantages such as extracellular secretion expression, stable expression, high production, easy purification and the multi-copies of extra gene can repeatedly integrating into the Pichia Pastoris genes, so it is suitable for high density fermentationand is a good expression system. As a new extra gene expression system, Pichia Pastoris have been given more and more attentions, which has the eukaryote characteristic and easily to operate. On the other hand, it contains introducing promotor, we can use methanol to strictly regulate its expression. As the extra proteins secrete into the culture medium, it is useful to maintain natural configuration and activity of the proteins, and as a result the purification is relative easily. The viability of Pichia Pastoris is high, and the culture medium is relative cheap, up to now the fermentation technique of Pichia Pastoris has completely developed. Using Pichia Pastoris as expression system, the biomass is high, and suitable for large-scale culture in medical industry. In order to improve the biological activity and curative effects of hGM-CSF, and decrease its dosage and side effects, we have mutated the O-glycosylation sites of 9-Ser and10-Thr. We found that the molecular weight of the product expressed by the mutant gene is the same as that of the no mutant and the product still has the immunogenicity and biological activity, yielding product about 100mg/L at flask scale. Our data showed that it is significant to further research the glycosylation and the modification after expression in Pichia Pastoris.
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