Preparation And Characterization Of The Recombination RGD-containing Peptide From Wild Silk Fibroin

silk fibroin is natural animal protein synthesized and secreted by silkworm, whichcontaining domesticated silk fibroin (synthesized and secreted by Bombyx mori) andwild silk fibroin (synthesized and secreted by Antheraea pernyi, Antheraea yamamai,Samia cynthia ricini, Dictyopoea japonica Moore，Fish line silkworm and Attacus atlasLinnaeus etal). The current researches focus on Bombyx mori silk fibroin and thosestudies have shown that Bombyx mori silk fibroin material can support a variety of celladhesion, proliferation and differentiation as a biological material. While the amino acidsequence of Antheraea pernyi and Antheraea yamamai silk fibroin distribute amounts ofRGD short peptides which can effectively promote cell adhesion and provide a bettercell function interface as extracellular matrix materials. To systematically research therelationship between sequence, structure and function of wild silk fibroin, especially toreveal the sequences closely related to cell function in the amino acid sequence of wildsilk fibrion, we designed the sequence from gene level, and used E.coli expressionsystem to express the motif or its block combination for rearch. In this paper, wecompleted the expression of the recombination RGD-containing peptide fromAntheraea pernyi (Antheraea yamamai) silk fibroin, mainly included the followingwork:Firstly, designed the gene sequence AY1which encoding amino acid sequeseGSGAGGRGDGGYGSGSS from Antheraea pernyi (Antheraea yamamai) silk fibroin,and doubled which to24times to obtain extension gene AY24using gene recombinationtechnology. Verified the construcion of genes were right by agarose gel electrophoresisand DNA sequencing.Secondly, the gene sequence AY1and the doubled sequences were inserted intothree series of expression vectors pcDNA3.1B, pET-30a(+) and pGEX-KG respectively,and expressed in E.coli BL21by IPTG induction. Verified the construcion of expressionvectors were right by agarose gel electrophoresis and DNA sequencing. The pGEX-KG expression vector was selected to express recombinant protein after comparisons andanalyses, and verified that the expression of recombinant proteins were successfullyobtained by SDS-PAGE electrophoresis and Western blotting.Thirdly, discussed the best expreesion conditions of the fusion proteinGST-(-RGD-)12and GST-(-RGD-)24which were expressed by pGEX-AY12andpGEX-AY24respectively, and purified the two kinds of fusion protein by affinitychromatography column. Qualitative detections by SDS-PAGE electrophoresis andWestern blotting showed: the E.coli BL21containing pGEX-AY12induced6hoursunder the0.5mmol/L IPTG, the expression quantity of fusion protein GST-(-RGD-)12reaches maximum value which is about45mg per liter bacterium liquid; the E.coliBL21containing pGEX-AY24induced6hours under the1.0mmol/L IPTG, theexpression quantity of fusion protein GST-(-RGD-)24reaches maximum value which isabout28mg per liter bacterium liquid.At last, obtained target peptides (-RGD-)12and (-RGD-)24by using thrombinprotease enzyme digestion fusion protein GST-(-RGD-)12and GST-(-RGD-)24.Measured that the isoelectric point of the target peptides (-RGD-)12is about8.5, and thetarget peptides (-RGD-)24is about8.0by Zeta potential measurement and two-dimensional electrophoresis analysis. The amino acid composition analysis showed thatthe expression of fusion protein GST-(-RGD-)12and GST-(-RGD-)24were correct inE.coli. And preliminary explorations on the secondary structure of target peptides(-RGD-)12and (-RGD-)24were measured by Circular Dichroism, the results showed thatthe two target peptides were mainly α-helix structure.