Revision as of 10:50, 21 October 2021

PepACS

Sequence and Features

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

Fig1. Colony PCR results of AOX1-α factor-curA-AOX1 Terminator, AOX1-α factor-pepACS-AOX1 Terminator and AOX1-α factor-DsbC-AOX1 Terminator transformed E.coli.

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.

Fig2. Colony PCR result of yeast after electroporation through electrophoresis.

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.

Background related to hair structure

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

Fig3. SDS-PAGE result of Laccase GS115 4CL LOX2 ACC pepACS DsbC+pepACS detecetion in the supernatant.

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

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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.

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The experimental results showed that the Chemical perm agent had the most obvious effect, while others didn’t have any obvious perming effect. We’ve considered 3 reasons why short peptides have no obvious effect:
Maybe the peptides didn’t get into hair cuticle
The time for short peptides to form disulfide bonds wasn’t enough
The reaction temperature is not enough.
So, we get another exploring experiment.
Time and Temperature:
(1)Drop a drop of 3M NaOH into 0.01% short peptides solution. [PH test paper: PH=10]
(2)Control experiment: drop a drop of 3M NaOH into UP water
(3)Both react at 50°for 1h.
Re-explore a better condition for short peptides perming hair
1)immerse 10 hairs in SDS 3 times to remove charges on hair surface.
2)Wash and put them in 0.01% short peptides solution
3)Control experiment: Up water, the subsequent processing steps are the same as 1)2)
4)both:50℃,1h
Perming hair stress experiment
We used different kinds of perms for hairs with 2 groups of different hair, and the hair strains will be damaged after perming. So we designed and conducted stress experiments to test the mechanics of the hair after perming. The figure below shows the experiment data, by which we can know that perming by short peptides will have very little damage to the hair, and short peptide perm damage is less than chemical perm. Moreover, the results of the damaged hair groups showed that the damage of short peptide perm is much smaller than that of chemical perm. Hence, we speculated that perming procedure would cause some damage to hair, but the use of short peptide perm could be used to repair the damaged hair.

Fig6 Healthy hair stress test results (Horizontal: the deformation length; Ordinate: pull force).

It is shown that the hair of the short peptide perm group changes in shape under constantly increasing pull force, and most hair strains can withstand more pull than hair permed by chemical perms. This suggests that short peptide perms do less damage to hair performance than chemical perms. According to the experimental results, we judge that the concentration of short peptides has little effect on the results.

Fig7 Damaged hair stress test results(Horizontal：the maximum length; Ordinate: pull force).

This set of data comes from a pulling test of damaged hair. It is shown in this figure that both sets of hairs that use short peptides for perm can withstand more pull than the hairs after chemical perm. And there isn't much difference between the results of short peptide group and that of the water group. We speculate that short peptides can by some means repair the damaged hair, which also portrays the excellent performance of short peptide perm that it can protect the hair and maintain the hair quality.