Study On Exogenous Expression And Spinnability Of Recombinant Keratin And Keratin Like Polypeptides

Keratin is derived from natural renewable resources such as feather,wool and human hair.It is cheap and has good biological characteristics,so it is widely used in biomedical research.Due to its high content of disulfide bonds and complex three-dimensional structure,the chemical conditions for extraction of keratin are usually harsh and cannot be scaled up in industry.In addition,the chemical method to extract keratin exist the following problems also make it difficult to application in the pharmaceutical industry: animal sources of keratin caused the immune tolerance and the risk of disease transmission across species,human hair keratin due to frequent with a sharp drop in quality,and from different sources or the same source nature of keratin significant difference between different batches.These problems have seriously hindered the progress of keratin-based materials research.The precise control of protein primary sequence,chain length and stereochemistry by DNA recombination technology can solve the deficiency of chemical preparation of keratin.In this paper,molecular biological methods were adopted to construct the recombinant plasmid through gene level molecular design,and the expression was induced by different host,further identification,separation and purification,and finally obtained the recombinant protein with customized sequence,which laid a good foundation for the subsequent spinnability research involving keratin.ELP is a class of classical genetically engineered recombinant peptide polymers derived from mammalian elasinogen.The composition of pentapeptide repeat sequence Val-Pro-GLy-Xa A-Gly（Xaa can be any amino acid except Pro）endods it with temperature-responsive reversible phase transformation properties.NCBI searched the sequences of type I and type II human hair keratin,analyzed bioinformatics and constructed HHK.By fusing ELP and HHK,the el P-HHK fusion was purified based on the temperature responsive reversible phase transition property of ELP.The recombinant plasmid p ET-28a（+）-ELP--HHK was specifically constructed by enzyme digestion and enzyme linking using E.coli BL21（DE3）as the host of heteroexpression.The recombinant plasmid was transferred into E.coli BL21（DE3）competent cells by heat shock transformation.Resistance screening markers were further used to identify positive colonies.Based on the negative regulation of T7 promoter induced expression type,ELP-HHK was induced by IPTG,and specific purification of ELP-HHK was attempted by reversible phase transition cycle.The results showed that the rich free sulfhydryl groups in human hair keratin polypeptide HHK formed cross-linking of hydrophobic core intermolecular disulfide bonds during the temperature-responsive phase transition of the fusion,resulting in irreversible phase transition even in the presence of reducing agents.In this paper,the exogenous expression conditions of another recombinant keratin FKeratin were optimized,and the induction temperature,induction time and concentration of inducer were optimized by Dot Blotting.The maximum expression of soluble recombinant keratin FKeratin was obtained under the conditions of 37℃ induction temperature,10 h induction time and 0.75 m M IPTG concentration.Bradford method detected the final FKeratin concentration of 41.97 mg/L fermentation broth and characterized its basic physical and chemical parameters.In order to find a suitable electrospinning process for recombinant keratin and keratin-like polypeptides,a preliminary attempt was made.The results show that when the foreign protein is alone,the fiber can not be formed by electrostatic spinning,but by blending with polymer,the fiber with good shape and smooth surface shape can be obtained.