Part:BBa_K3711016
α-factor
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 244
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
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, Pro-region 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.
Molecular cloning
First of all, we need to amplificated all the commercially synthesized plasmid to acquire enough amount for further study. After transformation, colony PCR is applied for confirmation. Then we go for plasmid extraction.
Bright bands of identical sizes from colony PCR result demonstrates that target plasmid had successfully transformed into E.coli
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
SDS-PAGE
After confirmation from colony PCR and sequencing, we using the successfully integrated yeast for expression. At first, we try to detect our target protein in the supernatant since there is signal peptide.
Due to glycosylation modification of yeast expression, the molecular weight exhibited on SDS-PAGE will be larger than theoretical. Primary detection shows that we have laccase, 4CL and ACC bands of about 75kDa, LOX2 band of 100+kDa and DsbC+pepACS of about 40kDa, all of which is a bit larger(Laccase：57.01 kDa; 4CL：61.88 kDa; ACC：63.40 kDa; LOX2：102.88 kDa; DsbC+pepACS：31.72 kDa) but still within explainable and acceptable range, which could be evidence of successful expression.
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