Construction And Expression Of Recombinant Protein From Bombyx Mori Silk Fibroin Heavy Chain

Bombyx Mori silk fibroin is a kind of natural animal protein increasingly regarded asbiomedical materials in the studying fields, especially the tissue engineering scaffolds,mainly applied in tissue repair materials. Repairing different organizations has differentrequirements for the structures and properties of materials. The structures and properties ofmaterials are decided by the protein secondary structure while the protein secondarystructure depends on the composition and sequence of amino acid. Silk fibroin consists ofFib-H, Fib-L and P25. Each sequence of silk fibroin has special meaning to itself, so wemust 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. In order to better study the relationship amongprotein sequence-structure-function, this thesis analyzed and designed the fibroin heavychain by using genetic engineering to reconstruct and clone the genetic sequence of thecrystalline and amorphous regions of fibroin heavy chain, and adopting microorganismEscherichia coli expression system to express and prepare a large number of protein, andthen pure it. And initially the expression products were made qualitative and quantitativeanalysis, which provided materials and methods for the study of structure and function(especially biological characteristic) effect of the recombinant bombyx mori silk fibroin.According to the genetic sequences of fibroin protein and the coding sequence ofamino acids, combining with the previous research of this topic, this thesis constructedseveral typical repeat peptides of crystalline domain (GAGAGX)16(X=A, S, V, Y) and anamorphous area peptides (F), crystalline domain (GAGAGS)16and different ratio ofamorphous peptides (Fn) and built the pGEX expression vectors. Using PCR to cloneC-termini coding genes of fibroin heavy chain and construct the cloned plasmid, laid thefoundation for further restructuring with fibroin heavy chain. Agarose gel electrophoresisand DNA sequencing revealed all cloned plasmids and expression vectors were correctwithout gene deletion or mutation.IPTGThe expression vectors were transferred in Escherichia coli BL21and added IPTG tothem to induce expression. SDS-PAGE electrophoresis and Western blot technique showed that expression vector pGEX can well express. This thesis mainly studied the expression ofFib-H gene recombinant between crystallization (GAGAGS)16peptides and different ratioof amorphous peptides (Fn). The research focuses on expression vectors pGEX-gs16f1,pGEX-gs16f4, pGEX-gs16f8and pGEX-gs16f12, whose corresponding products wereGST-GS16F1, GST-GS16F4, GST-GS16F8and GST-GS16F12fusion proteins with aGST-tag. And then the author optimized the expression conditions of four fusion proteinsand investigated the effect of different inducer (isopropyl-β-D-thiogalactoside)concentration (0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0mmol/L) and different inductiontime (0,1,2,3,4,5,6,7,8h) on the expression of the three fusion protein. The results revealedthat the IPTG concentration of the three fusion proteins is0.2,0.1,0.4and0.6mmol/L,and induction time is3,4,6and3h, respectively.A large number of three kinds of fusion proteins GST-GS16F1, GST-GS16F4andGST-GS16F8were expressed and the high purity fusion proteins were collected by usingthe GST affinity chromatography column and SDS-PAGE electrophoresis. The proteinconcentration was measured by ultraviolet absorption method. The results showed that thethree fusion proteins production can be about53.20,30.59and14.02mg/L.After purification and ultrafiltration of fusion protein GST-GS16F1, GST-GS16F4,GST-GS16F8and target peptides GS16F1, GS16F4, GS16F8which obtain after usingthrombin enzyme isolated from. From the mass spectrometry of molecular weight of thepurified fusion protein and target peptides, the measured results were consistent withtheoretical values.The research analyzed the property of isoelectric points and amino acid compositionsof the three kinds of purification and ultrafiltration fusion proteins GST-GS16F1,GST-GS16F4and GST-GS16F8. The result showed that the isoelectric point ofGST-GS16F4and GST-GS16F8was between4and4.5, and that of GST-GS16F1wasbetween5and5.5by the Zeta-otentiometer, which corresponded to the theoretical values.The actual measured value of amino acid composition analysis is also consistent with thetheoretical value. These results indirectly illustrat the right expression of the recombinantproteins.