BTP-1M,Targeted TachyplesinⅠpolymer

Tachyplesin Ⅰ(TP-Ⅰ)is a cysteine-rich antimicrobial peptide derived from the hemocytes of horseshoe crabs.The TP-ⅠM is composed of four TP-Ⅰs.The TP-ⅠM containing 73 amino acids was joined by E, cleavage sites of Glu-C. BTP-ⅠM is a sequence based on the specific targeting of the signal molecule CSP in the quorum sensing signal system of Streptococcus mutans.The gene sequence that can bind to the cell membrane protein of Streptococcus mutans, and the antimicrobial peptide linked to this sequence will increase the killing effect of other antimicrobial peptides of the same class on Streptococcus mutans.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 28
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

2020 SZPT-CHINA

Construction of the antimicrobial Peptides Multimer(BTP-ⅠM)

In order to achieve the expression of BTP-Ⅰ, we used E as the connection site to form a specific targeting antimicrobial peptide polymer (BTP-ⅠM). The amino acid sequence of BTP-ⅠM is shown in Fig.1.

Fusion expression of BTP-ⅠM in E. coli system

Construction and Identification of Recombinant Expression Vector of antimicrobial peptides

We cloned the BTP-ⅠM gene into the pGEX-4T-2 expression vector. Positive clones were selected from LB plates containing 100μg/mL ampicillin for restriction analysis. The results show that the fragment size is the same as BTP-ⅠM, shown in Fig.2. Hence, the recombinant expression vector pGEX-4T-2-BTP-ⅠM was successfully constructed.

Expression of GST-BTP-ⅠM in E. coli BL21

The recombinant strain E. coli BL21 (DE3) with pGEX-4T-2-BTP-1M vector was induced by IPTG for 6 hours and then collected for SDS-PAGE and Western blot analysis. The results of SDS-PAGE were shown in Fig.3.

After IPTG induction, the E. coli BL21 transferred with pGEX-4T-2 vector produced 26kDa GST protein and E. coli BL21 transferred with pGEX-4T-2-BTP-ⅠM produced 44kDa protein matched well with our GST-BTP-ⅠM fusion protein.In contrast, Non-induced E. coli BL21 cells did not express GST or GST-TP-ⅠM.Western blot results showed 44kDa fusion protein carry GST tag. The results showed that the constructed tandem BTP-ⅠM could be expressed in the fusion expression system.

Fusion protein purification results

Recombinant bacteria were induced to express, and the cells were collected, ultrasonically broken, and the inclusion bodies were dissolved. After affinity chromatography, the fusion proteins GST-BTP-ⅠM was obtained. The SDS-PAGE electrophoresis detection result is shown in the Fig.4.

Reference

[1]Zhou L, Liu Z, Xu G, et al. Expression of Melittin in Fusion with GST in Escherichia coli and Its Purification as a Pure Peptide with Good Bacteriostatic Efficacy[J]. ACS omega, 2020, 5(16): 9251-9258.

[2]Jin Yuanbao,Wang Ying,Liu Liping,Wang Qian,Jin Gang,Zhang Lijun,Dai Jianguo.Study on the effect of Limulus I on Proteus vulgaris[J].Biotechnology Bulletin,2014(05):190-196.