Coding

Part:BBa_K3196099:Experience

Designed by: Hao Qiu   Group: iGEM19_HUST-China   (2019-10-16)
Revision as of 12:29, 10 October 2021 by JunfengHui (Talk | contribs) (→‎Applications of BBa_K3196099)


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===Applications of BBa_K3196099=== α-factor signal peptide is a leading peptide of mating factor 1 (MF1) n-terminus secreted by yeast α cells. It consists of 86 amino acid residues, including pre-peptide (pre-sequence) and pro-region (pro-region) sequences.Studies have shown that α-factor signal peptides play an important role in the secretion of exogenous proteins in Pichia pastoris, and the three N-glycosylation of pro-region is important for the secretion of peptides. We improved the structure and function of α-factor and explained the processing of α -factor-guided exogenous protein in the secretion pathway, which helped us to deepen our understanding of α-factor. Meanwhile, we summarized the endogenous signal peptide expressed by exogenous protein in Pichia pastoris.This will facilitate the expression and optimization of pichia pastoris α-factor endogenous signal peptide in the future. We compared the difference in the expression of exogenous protein guided by s. cerevisiae α-factor signal peptide with n-glycosylation modification and without n-glycosylation modification. The linearized recombinant plasmid P PIC9KS.The recombinant GS115S. Ce-n23/57/67q-egfp was obtained by electric transfer, and cultured together with GS115S. Ce-egfp for 24h BMGY. Under the same conditions (OD=1, volume: 5ml), the supernatants were harvested for 72h after methanol induction, and western-blot analysis was performed. The results showed that EGFP secretion was decreased by 50.3% after the mutation of N to Q at three potential glycosylation sites of S.cerevisiae α-factor signaling peptide.The results showed that s. cerevisiae α-factor signal peptide was modified by N-glycosylation when expressed in Pichia pastoris, and n-glycosylation of s. cerevisiae α-factor signal peptide contributed to the improvement of exogenous protein production.

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