Part:BBa_K3711051
AOX1-α factor-DsbC-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1187
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1342
- 1000COMPATIBLE WITH RFC[1000]
Description
This is a composite component for expressing DsbC outside the cell. DsbC is transcribed and translated into protein disulfide isomerase(PDI)，which is the key enzyme for straightening. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, DsbC is translated and excreted from the cell.
Usage and Biology
DsbC is one of five essential protein needed for forming disulfide bonds in E.coli, functioning as disulfide bond isomerase during folding process of oxidized protein in periplasmic space. DsbC is a homodimer of size of 2x23kDa, with enzymatic activity of protein disulfide bonds isomerase and molecular chaperone.
The crystal structure of DsbC shows that disulfide bonds are formed in all Cys-X-X-Cys reaction center. Through the hinge, which allows the reaction center moving relatively, a single thioredoxin-like domain is linked to dimer domain in the amino-terminal. The widely-existing uncharged seam between reaction center may participate in peptide combination and enzymatic reaction of DsbC folding enzymes.
Linking DsbC with peptides rich in cysteine, we build a transporting and auxiliary binding complex with disulfide bond isomerase and short peptides.
DsbC is a periplasmic disulfide isomerase of Gram-negative bacteria. Its activity is only 30% of that of eukaryotic disulfide isomerase (PDI) isomerase and mercaptan oxidoreductase. However, DsbC showed more significant chaperone activity than PDI in promoting reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vitro and inhibiting aggregation during renaturation. Like PDI, DsbC is a disulfide isomerase with chaperone activity, but it may recognize different folding intermediates.
Studies have shown that PDI is linked to polypeptides containing cysteine, which can help polypeptides bind to hair and help repair hair damage. In the process of hair straightening, we need to replace the original peptides, while DsbC is small enough to enter the hair to help the new peptides bind to the hair.
Molecular cloning
Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.
The bands of AOX1-α factor-curA-AOX1 Terminator (almost 3000bp), AOX1-α factor-pepACS-AOX1 Terminator (almost 2000bp) and AOX1-α factor-DsbC-AOX1 Terminator (almost 3000bp) from colony PCR are identical to the theoretical lengths of 2875bp, 1987bp and 2722bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these 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 DsbC+pepACS of about 40kDa, which is a bit larger than 31.72 kDa but still within explainable and acceptable range, which could be evidence of successful expression.
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