Preliminary Study Of Glycosylation In Pichia Pastoris

P. pastorisis is a methylotrophic yeast that had already been used for production of a wide variety of heterologous proteins. Besides the advantages of protein processing, folding and post-translational modifications in eukaryotic cells, it offers the benefits of cost-effective and easy scale-up in E. coli. Additionally, it does not secrete too much intrinsic protein. This makes P. pastorisis as an excellent candidate for the production of protein-based therapeutic agents. Though series of profits come from pichia pastoris expression, but it is not suitable to express glycan proteins due to presence of yeast-specific outer-chain mannosylation. P. pastorisis N-glycans of the high mannose (Man) type out chain biosyntheses in yeast are initiated by a 1,6-mannosyltransferase encoded by och1 gene in P. pastorisis. Much work had already been done in och1 gene knokout in P. pastorisis, but there are still some problems during the genetic manipulation. Normally double homologus recombination was used to knockout och1, but it is highly inefficient. In this research, two steps of genetic recombination strategy was used to knockout ura3 and och1.Two steps of genetic recombination knock out target gene efficiently. The method is that knocking out open reading frame first, then linking this fragment to the vector with screening marker. The first recombination is to input the plasmid into the yeast, checking whether the integration site is right, and the second is to knock out the target gene. At the same time, screening marker is removed.The ura3 gene of GS115 was knocked out through this technology. The mutant of this strain cannot grow in the medium lack of uracil, but after adding uracil, grows well and goes down to the later generation stably. GS115 with ura3 mutant leads to additional selecting marker in glycosylation engineering and can be used as the initial strain for further research.The same recombination method was used to remove och1 gene of JC308. In addtion, we also characterized the physical type of X-33 och1 mutant strain. The och1 gene is responsible for the high mannose of glycoprotein. Comparisoning growth and phenotype of both Pichia pastoris X-33 wild strain and X-33 och1 mutant strain, to lay foundation for further research. Compared with wild strain, the characteristic of och1 mutant lies in as follows: slow growth, decreased biomass, sensitive to temperature, which can be counterbalanced by the addtion of osmotic pressure, unabled to grow in culture containing congo red, the cell wall deficient in division, insufficient cell vitality, high death rate in cultivation process.