The Duke University team submitted the Tachyplesin-I (TP-I) (BBa_K1835501) part in 2015. TP-I is a cysteine-rich antimicrobial peptide derived from the hemocytes of horseshoe crabs. It is a broad-spectrum antibacterial peptide with good antibacterial function.
But Duke University did not express TP-I in 2015, only tested the antibacterial effect of TP-I on E.coli.
This year, we conducted a specific data characterization of TP-I. We measured the MIC of TP-1 against Streptococcus mutans, and the results are shown in Figure 1. The MIC value of TP-1 against Streptococcus mutans was 8μg/mL, indicating that TP-1 has a good inhibitory effect on Streptococcus mutans.
Figure 1．TP-1 inhibits MIC of Streptococcus mutans
Meanwhile，we adopted the strategy of tandem fusion expression of TP-I to obtain high-yield TP-I by considering the difficulty of short peptide expression in engineering bacteria. The low fusion expression yield of single peptide facilitates the subsequent use of GST tags. The high purity peptide was obtained by enzymatic hydrolysis for analysis after protein purification.
The GST-TP-1M were transferred into E.coli BL21 and then selected transformants with ampicillin. The transformants were cultured in LB broth and induced by IPTG. Then, the bacteria were collected into new microtubes and break the walls. The fusion protein was separated by GST-labeled affinity column chromatography and then verified by SDS-PAGE and Coomassie brilliant blue staining.
Figure2. SDS-PAGE analysis of expression and purification of GST-TP1M in E.coli BL21
M, Marker;Lane 1, E.coli BL21 (pGEX-4T-2-TP-1M) without IPTG inducing; Lane 2, E.coli BL21 (pGEX-4T-2-TP1M) with IPTG inducing; Lane 3, Broken supernatant stock solution; Lane 4, Effluent; Lane 5, Rinsing liquid; Lane 6, Eluent
After expressing TP-1 in tandem fusion, it is convenient for GST-labeled affinity column chromatography. The GST tag is an antibody recognition site, and there are many prepared antibodies on sale. Once we have this label, we can perform Western Blot and other techniques based on antigen-antibody reactions.
Reference
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