Coding

Part:BBa_K3196099:Design

Designed by: Hao Qiu   Group: iGEM19_HUST-China   (2019-10-16)
Revision as of 14:45, 5 October 2021 by JunfengHui (Talk | contribs) (→‎Design Notes)


α-factor


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 34
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 34
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
    Illegal XhoI site found at 255
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 34
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 34
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

signal peptides Structure and function of A-factor signal peptide 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.

Source

signal peptides

References