Part:BBa_K3711014
DsbC
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 126
- 1000COMPATIBLE WITH RFC[1000]
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.
Molecular cloning
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.
Biologically speaking, human hair is one kind of complex fibers with various of shapes and components. The main ingredient of the hair is keratin, which is a bright structural protein, as a member of intermediate filament superfamily. According to calculation of amino acids constituent, 18% of keratin is cysteine. The hair filament is consisting of three main layers: cuticle, cortex and medulla.
The medulla locates in the core area of the hair, which is partly or totally deficient in slender ones.
The cortex is the main part of the hair, which consists of huge fiber of intermediate filaments. The helical α-keratin, major part of the intermediate filament, provides mechanic sustaining force. Single fiber in cortex cells is separated by membrane consists of KAPS, which is rich in cysteine but unclear of space structure. The supplementary fiber of keratin is entangled by iron force, hydrogen bonds, Van der Waal’s force and disulfide bonds.
Wrapping outside of the cortex, the cuticle is consist of layers after layers of cells, constructing a kind of squama-like structure. Every cuticle cell has respective lower filaments layer, whose protein is entangled by cysteine.
SDS-PAGE
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 DsbC+pepACS of about 40kDa, which is a bit larger(DsbC+pepACS：31.72 kDa) but still within explainable and acceptable range, which could be evidence of successful expression.
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