Preparation And Structure Analysis Of Recombinant Protein From Bombyx Mori Silk Fibroin Heavy Chain

In recent years,silk fibroin have been increasingly regarded as biomedical materials,especially the tissue engineering scaffolds.Tissue repair materials have different requirements for the structures and properties of materials.The structures and properties of materials are decided by the protein secondary structure while the protein secondary structure depends on the composition and sequence of amino acid.Silk fibroin consists of Fib-H,Fib-L and P25.Each sequence of silk fibroin has special meaning to itself,sowe must find out the scientific problems.The Fib-H,as a major component of fibroin,composing of crystalline and amorphous region staggered,ordered and neat structures,which is helpful to reasonable design.This paper focuses on the preparation and structure of the repective sequence and different ratio of non repective sequence.This paper mainly used Escherichia coli expression system to express the repective sequence and different ratio of non repective sequence,after separation and purification and thrombin digestion,the target peptides constructed by repective sequence and different ratio of non repective sequence from Fib-H were obtained.The correctness of the target peptides after thrombin digestion was indirectly characterized by isoelectric point determination.On this basis,the changes of secondary structure of target peptide after treatment with different external conditions were analysed.（1）Optimized the expression of non-repeat region gene expression vectorThe already constructed pGEX-f（1）、pGEX-f（4）and pGEX-f（8）containing non-repeat gene GS16f（1）with polypoid GS16f（4）and GS16f（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 investigated the 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-GS16F（1）was high when the initial bacterial density OD₆₀₀=1.2,the induction time was 1 hour,was about 78.9 mg with a liter bacterium solution;while GST-GS16F（4）was highly expressed about 52.6mg every liter bacterium solution in the case that initial bacterial density OD600=1.5,the induction time was3 hours;besides,the expression level of GST-GS16F（8）was 28.3mg with a liter bacterium solution when the initial bacterial density OD600=1.8,the induction time was 1 hour.（2）Purification of the expression products and the release of the target peptideHigh purity fusion proteins were obtained by using GST affinity chromatography column.The GST tag was removed by thrombin and the target peptide GS16F（1）,GS16F（4）and GS16F（8）were separated and prepared.（3）Characterization of expression productsThe target peptide were 4.14,3.82 and 2.98 consistent with the theoretical value of3.77,3.16 and 2.87.These data showed the target peptides were right.（4）Preliminary exploration on the secondary structure of target peptidesThe secondary structure of target peptides was measured by CD spectroscopy.The CD spectroscopy and Raman spectroscopy results showed that three target peptides were predominantly in the structure ofβ-fold.The CD spectra showed that as the molecular chain grows,the molecular conformation changed,The content ofβ-folded in the molecular conformation decreased with increasing non-repetitive sequence in the molecular chain.（5）Analysis of secondary structure of target peptide at different external conditionsThe conformational changes of the peptides at different temperatures（4℃,20℃,37℃,60℃）for 24 hours were investigated.Secondly,the conformational changes of the target peptide solution were investigated under the influence of pH value（pH2,pH3,pH4,pH5,pH6,pH7.4,pH8,pH9 and pH10）.Finally,the effects of different concentrations（50mM,100mM,150mM,200mM,250mM,300mM and 400mM）of different ion（Na⁺and K⁺）on the secondary structure of the target peptide were investigated.