Difference between revisions of "Team:TCU Taiwan/Project/Experimental"

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為了能夠有效的為了能夠使我們更有效地拿到我們的指定抗菌肽，我們在設計序列時在AMPs的N-terminal加了一個signal peptide。這個signal peptide是來自於<I>S.lividans</I>的幾丁質酶Ｃ(signal peptide sequence: MGFRHKAAALAATLALPLAGLVGLASPAQA)。在進行轉錄後，signal peptide會引導AMPs到E.coli的分泌系統。在pre-mature peptide穿越periplasmic space，peptidase會辨認signal peptide上的Ala-Gln-Ala氨基酸序列，並且在signal peptide和mature peptide連接處的兩個Ala之間將之分開。最後分泌出AMPs到LB 培養液中。<BR>
 
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為了確保我們的分泌系統能順利運作。我們調整了Signiferin 還有Epinicedin-1的N-terminal加了一個Ala去幫助peptidase能夠順利做切割，並且接續將我們的AMPs分泌到胞外。我們在網頁的modeling page中呈現了用軟體預測結構的結果，分析在我們加了一個Ala後是否會影響蛋白的摺疊過程。
 
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In order to have more efficient to get our AMPs, we treated signal peptide upstream of the N-terminal of antimicrobial peptides to facilitate the peptide production. This signal peptide is comes from chitinase C of <I>S.lividans</I> (MGFRHKAAALAATLALPLAGLVGLASPAQA).<a href="#tcu_sing_references_1">[1]</a> After translation process signal peptide will lead AMPs to the secretion system of <I>E.coli</I>. <a href="#tcu_sing_references_2">[2]</a> When the pre-mature peptides go through the periplasmic space, peptidase will identified the cleavage site Ala-Gln-Ala and cut at the double Ala at the signal and mature peptide linkage site. Then separate signal peptide from AMPs. Finally, secreting AMPs to the LB culture medium.
 
In order to have more efficient to get our AMPs, we treated signal peptide upstream of the N-terminal of antimicrobial peptides to facilitate the peptide production. This signal peptide is comes from chitinase C of <I>S.lividans</I> (MGFRHKAAALAATLALPLAGLVGLASPAQA).<a href="#tcu_sing_references_1">[1]</a> After translation process signal peptide will lead AMPs to the secretion system of <I>E.coli</I>. <a href="#tcu_sing_references_2">[2]</a> When the pre-mature peptides go through the periplasmic space, peptidase will identified the cleavage site Ala-Gln-Ala and cut at the double Ala at the signal and mature peptide linkage site. Then separate signal peptide from AMPs. Finally, secreting AMPs to the LB culture medium.
 
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Revision as of 16:35, 15 September 2015



Signal Peptide

In order to have more efficient to get our AMPs, we treated signal peptide upstream of the N-terminal of antimicrobial peptides to facilitate the peptide production. This signal peptide is comes from chitinase C of S.lividans (MGFRHKAAALAATLALPLAGLVGLASPAQA).[1] After translation process signal peptide will lead AMPs to the secretion system of E.coli. [2] When the pre-mature peptides go through the periplasmic space, peptidase will identified the cleavage site Ala-Gln-Ala and cut at the double Ala at the signal and mature peptide linkage site. Then separate signal peptide from AMPs. Finally, secreting AMPs to the LB culture medium.

To make sure the secretion system is work we attached an Ala at the N-terminal of AMPs. So we had modified the amino acid sequence of Signiferin and Epinecidin-1 to facilitate the secretion process. And we had used structure prediction software to analysis the attached Ala affect the peptide folding process or not in our modeling page.

References


[1] Fujii, T. and K. Miyashita, Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans. J Gen Microbiol, 1993. 139(4): p. 677-86.

[2] Tokuyasu, K., et al., Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells. FEBS Lett, 1999. 458(1): p. 23-6.





             
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