Preparation And Effect On Cells Of Recombinant Silk Fibroin Non-repeat Region Peptides

B. mori silk fibroin is a natural animal protein fiber with remarkable mechanical properties and biocompatibility. It is increasingly used as a tissue repair material, Different tissues have different requirements on the structure and performance of the material. especially the amino acid alignment of protein has a decisive influence on its biological properties. So it is very important to study the relationship between the composition and structure of silk fibroin and its effect on cell. B. mori silk fibroin is mainly composed of heavy chain, light chain and P25 protein with a mole ratio of 6:6:1. The heavy chain is an important part of silk fibroin, which is composed of N-terminal, middle core area and C-terminal. The core area includes the repetitive and non-repetitive regions(Hereinafter referred to as non-repetitive region). In order to obtain a clear various parts of the peptides, genetic engineering is one of the advanced technological means. This paper focused on the characteristics of the non-repetitive sequences.The non-repeat region peptide expression system has been constructed by our laboratory, on this basis, this paper mainly used Escherichia coli expression system to express the non-repeat region fusion protein, after separation and purification and thrombin digestion, the non-repeat regiontarget peptides were obtained. Identified the correctness of the fusion protein and the target peptides after thrombin digestion by using mass spectrometry analysis and amino acid composition analysis and isoelectric point determination. The secondary structure of the target peptide was analyzed, and the effect of the target peptide on the growth of endothelial cells was studied.(1) Optimized the expression of non-repeat region gene expression vector.The already constructed p GEX-f(1)、p GEX-f(4) and p GEX-f(8) containing non-repeat gene f(1)with polypoid f(4) and f(8) were transformed into Escherichia coli BL21, and induced by IPTG to get the fusion proteins GST-F(1)、GST-F(4) and GET-F(8).The expression of the three kinds of fusion proteins were first determined by SDS-PAGE. Then further investigatedthe factors of expression level including initial bacterial density, the concentration of IPTG and induction time. The results of SDS-PAGE showed that the expression level of GST-F(1) was high when the initial bacterial density OD600=1.5, the IPTG concentration was 0.1 m M and the induction time was 1 hour, was about 58.8 mg with a liter bacterium solution; while GST-F(4) was highly expressed about 39.2mg every liter bacterium solution in the case that initial bacterial density OD600=1.2, the IPTG concentration was 0.2m M and the induction time was 4 hours; besides, the expression level of GST-F(8) was 26.6mg with a liter bacterium solution when the initial bacterial density OD600=1.5, the IPTG concentration was 0.1m M and the induction time was 1 hour,.(2) Purification of the expression products and the release of the target peptide High purity fusion proteinswere obtainedby using GST affinity chromatography column. The GST tag was removed bythrombin and separated target peptide. The released target peptideswere highly purity indicated by SDS-PAGE. The obtained fusion proteins and target peptides were later analyzed with mass spectrometry. the test value of fusion protein GST-F(1), GST-F(4) and GST-F(8) were 31.5, 43.8 and 59.0k Da, consist with their theoretical value of 31.0, 43.0, 58.9k Da. the measured values of F(1), F(4) and F(8)were 4.8, 16.8 and 32.8k Da also agreed well with the theoretical values of 4.8, 16.8 and 32.8k Da.(3) Characterization of expression productsThe amino acid composition analysis of the obtained fusion proteins and target peptide showed the measured amino acid composition values were constant with theoretical values. Isoelectric points of fusion protein and target peptide results revealed that the measured isoelectric points of fusion proteinwere 4.3, 3.6 and 3.4, consist with their theoretical value of 5.35, 4.56 and 4.22, the target peptide were 3.3,3.2 and 3.0 consistent with the theoretical value of 3.7, 3.2 and 2.9. These data showed the obtained fusion proteins and target peptides were right.(4) Preliminary exploration on the secondary structure of target peptidesThe secondary structure of target peptides was measured by FTIR and CD spectroscopy. The FTIR results showed that F(1) form mainly random coils, whereas F(4) and F(8) have appeared random coils and α-helices structure. While the CD revealed that the molecular conformations changed with the molecular chain lengthening, from random coils(F(1)) to α-helices(F(8)).(5) Research on hydrophilic modification of polyester film and the growthof endothelial cellsby target peptide.The Coomassie Blue Staining, XPS and static water contact angle tests proved that target peptide F(1)、F(4) and F(8) could be grafted to PET films, and the maximum grafting amounts was 0.020, 0.008 and 0.006μmol/sheet(diameter=1.5cm), the corresponding water contact angle can be decreased from 77° to 32°,35° and 39° respectively. Study on HUVEC proliferation of the PET flims grafted with F(1), F(4) and F(8) indicated that F(1), F(4) and F(8) were benefited to the endothelial cells growth,when the grafted amount of 0.015,0.006 and 0.004 mol / sheet respectively can significantly increase the growth of endothelial cells.