Revision as of 06:36, 20 October 2021

α-factor

   This signal peptide is a signal peptide carried by Pichia pastoris itself. In this project, we compared the enzyme activity expressed by it with that expressed by other signal peptides to prove the expression ability of different signal peptides to enzyme activity.
   At present, the most widely used and most successful signal peptide in Pichia pastoris system is saccharomyces cerevisiae A-factor signal peptide, so its structure and function have been studied in detail.α-factor signal peptide is a leading peptide of MATING factor A (MF1) at the n-terminus of mating factor 1 (MF1) secreted by yeast A cells, and its encoding gene is located on the chromosome XV1 of S. cerevisiae.α-factor signal peptide consists of 86 amino acid residues, and actually contains pre-peptide (pre-sequence) and pro-region (pro-region) sequences.Amino acid residues from 1 to 19 were pre-sequence, and residues from 20 to 86 were pro-region.The pre-sequence is divided into three functional regions: N-region(1-6), H-Region (7-15), and C-Region (16-19).N-region is a positively charged polar amino acid residue;H-region is a hydrophobic amino acid sequence, and its hydrophobic A-helix structure can effectively promote the transport of new peptides.C-region consists of conserved neutral amino acids and can be cleaved by type I signal peptidase.The first six amino acid residues of n-terminus of pro-region are evolution.
   The conserved output signal 6 peptide APVNTT, which are characterized by hydrophobic aliphatic chain amino acids.Pro-region is the most studied part at present, because it has 66 amino acid residues, indicating that it has certain structure and function.Pro-region consists of one A-helix and five β folds. If all amino acid residues in the range of 57 ~ 60 are deleted, the structure of pro-Region will be changed and its function will be affected.
   In addition, studies in S. cerevisiae found that there were three N-glycosylation sites (N23,N57 and N67) on the pro-region. Elimination would lead to a decrease in a-factor secretion but would not disappear, suggesting that N-glycosylation is important but not necessary for peptide secretion through the pathway.Pro-region glycosylation may contribute to the improvement of secretion efficiency, and studies have shown that moderate glycosylation contributes to the expression of foreign proteins.
   In addition, Proregion itself has a tendency to self-aggregate, and N-glycosylation can prevent pro-region from focusing in the endoplasmic lumen, thus improving secretion efficiency.Pro-region also acts as molecular chaperone to improve efficient folding of the guided protein,Endoplasmic reticulum associated degradation (ERAD) can be transported to proteasome for degradation.

Characterization

Sequence and Features