Expression,Gene Modification And Hemostasis Of Recombinant Human Hair Keratin K37-3?

With the increasing demand for rapid hemostasis in clinical and emergency situations,the design and development of new hemostatic dressings has become a hot research topic.As a natural biological material,human hair keratin has the advantages of excellent hemostatic effect,wound repairability,biocompatibility and degradability,and it was widely used in the field of biomedicine.In view of the limitations of the complex,quality and amino acid composition of human keratin extracts,our group has carried out recombinant expression and purification of human hair keratin K37,and proved for the first time that K37 recombinant protein also has good hemostasis performance.However,the region of effective hemostatic function in the human hair keratin K37 sequence is not clear,and since keratin is naturally insoluble,recombinant keratin is also an insoluble inclusion body.Therefore,studying the hemostatic function region of human hair keratin and improving the solubility of recombinant keratin through structural modification are of great significance for the development and design of novel hemostatic protein materials.In this study,we extracted the third ?-helix structure,the K37-3? sequence,which has the longest sequence in recombinant keratin K37 and contains the most comprehensive amino acid information.In order to improve the solubility of keratin,the CMK37-3? sequence was obtained by genetic modification technology.Then,the recombinant proteins of K37-3? and CMK37-3? were expressed,purified and characterized by recombinant protein heterologous expression technology.Finally,a new hemostatic dressing-recombinant keratin/polycaprolactone fiber composite membrane was prepared by electrospinning and coating method to investigate the hemostatic properties of K37-3? and CMK37-3? recombinant keratin.The main research contents and conclusions are as follows:(1)By intercepting the third ?-helix in the keratin K37 sequence,K37-3?.In addition,the recombinant protein was expressed and purified by the heterologous expression technology,and the physicochemical properties were characterized.The average yield of K37-3? after lyophilization was 120.22 mg/L,while the water-soluble fraction was only 6.496 mg/L.In addition,analysis by bioinformatics analysis,SDS-PAGE,MALDI-TOF-MS,UV-vis,FT-TR,CD and XRD means that: The target band of K37-3? was clear and single,the concentration was high,and the expression product was an insoluble inclusion body,and its molecular mass was 16.339 KD.Moreover,K37-3? also had the ultraviolet and infrared characteristic absorption peaks of the protein itself,the basic chemical structure was very similar to K37,and its secondary structure contained only ?-helix.At the same time,K37-3? was also present in the form of an amorphous structure.(2)In order to meet the functional requirements of clinical hemostasis,the K37-3? sequence was molecularly engineered,and the modified new sequence,CMK37-3?,was expressed,purified and physicochemically identified.The average yield of CMK37-3? powder after lyophilization was 36.18 mg/L,and the average yield after dissolution was 29.43 mg/L.It could be seen from the above physical and chemical characterization that the target band of CMK37-3? recombinant keratin was single clear and had high purity,and its molecular mass was 17.459 KD,and the water content of the product was excellent.In addition,compared with CK37-3? recombinant keratin,the structure and physicochemical properties of keratin were not changed before and after transformation.(3)After the K37-3? and CMK37-3? recombinant keratin were dissolved in 30% formic acid,the recombinant keratin/polycaprolactone nanofiber composite membrane was prepared by electrospinning technique and coating method.Then,the fiber composite membrane was tested by SEM,FT-TR,XRD,tensile elongation and contact angle analysis,and the hemostatic properties of the fiber composite membrane were investigated by in vitro and in vivo coagulation experiments.The results show that in the electrospinning process,when the mass fraction of PCL solution was 10%,the spinning was the smoothest,the obtained fiber was the most uniform,and the average diameter was 439.89±27.54 nm.In the coating process,when the keratin concentration was 2 mg/mL,the coating effect was the best,and there was no chemical reaction between the recombinant keratin and the PCL fiber membrane,but a hydrogen bond formed.At the same time,the crystallinity of the fiber composite membrane decreased,indicating that the two have good compatibility under the condition of formic acid as a solvent.Moreover,its hydrophilic properties increased,while the Young's modulus value decreased,but it was also comparable to the mechanical properties of other polycaprolactone/natural polymer based fiber composite membranes.In vitro procoagulant experiments found that recombinant keratin could increase hemoglobin adhesion,promote platelet activation and aggregation,and activate a series of cascades of enzymatic reactions.Simultaneously,K37-3? and CMK37-3? recombinant keratin/PCL composite membrane could significantly shorten the bleeding time and reduce the amount of bleeding in the model of hepatic and femoral artery injury in SD rats.,and the CMK37-3? fiber composite membrane had the best effect.(4)The cell compatibility of the hemostatic dressing was examined by inoculating L-929 cells on the surface of the recombinant keratin/polycaprolactone fiber composite membrane.The means of detection mainly include MTT,Calcein-AM/PI double staining and SEM.The results showed that K37-3? and CMK37-3? recombinant keratin/PCL fiber composite membranes were not toxic to cells and had good cell compatibility.Especially the addition of CMK37-3? recombinant keratin could further promote cell growth and proliferation.In summary,the third ?-helix fragment of human hair keratin K37 is an effective hemostatic function region of K37.Moreover,it has greatly improved the solubility of K37-3? recombinant keratin after molecular modification,and significantly improved its hemostatic effect.This study laid a theoretical foundation for the study of the hemostasis function of the other two ?-helices in K37 and the exploration of key sites for hemostasis,and provided new ideas for the development of new hemostatic drugs.