Part:BBa_K3711013
PepACS
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
PepACS consists of several short peptides(PepA, PepC, SPB) with flexible GS-linkers. All these short peptides are coded by genes of keratin from human hair and related protein, which enables PepACS to interact with keratin.
AAs seq of PepA: CCQSSCCKPSC
AAs seq of PepC: PIYCPPTCYH
AAs seq of SPB: LCRALIKRI
PepA is hydrophobic (less than 10%). Polarity interactions, for example, could be formed due to reactional groups in its sequence, which enhance its specificity and oriental binding ability. Peptides rich in cysteine may form covalent bonds with keratin in the hair, while some of it could recombine or even break the disulfide bonds of keratin. Cysteine could binds to sulfydryl, which could apparently reduce the disulfide bonds in keratin. When electron receptors, e.g. oxygen dissolved in solution, are available, cysteine residue could form disulfide bonds with them, which are oxidized from the butyl of cysteine.
The break down and reform of disulfide bonds is tightly related to the shape of the hair. Instead of alkaline and Sulphur containing relaxers, this peptide thought could be one brand new method of environmentally friendly control of hair shape.
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
There are irregular staining bands on the bromophenol blue line in the PepACS swimming lane. Although the molecular weight is not accurate, the successful expression can be judged according to the detection method we designed.
Experiment of short peptide perm
Short peptides perming hair experiment
We used 0.1% and 0.01% short peptides for perm experiment, and set chemical perm and up water perm as control group. The perming effect of short peptide is more obvious than that of ultra-pure water, which shows that in alkaline and 50° conditions, the hair cuticle is opened and the longer react time it takes, the better perming result we get. And after a series experiment, we found that hair dealt with SDS can have more obvious curl effect.
| Chemical perm agent(bought online) | Ultra-pure water |
| None |