Part:BBa_K3487013
Tachyplesin I multimers with GST-tag
The GST-tag has the size of 220 amino acids (roughly 26 KDa). It is fused to the N-terminus of the Tachyplesin I multimers BBa_K3487001 and includes a PreScission Protease domain for cleavage of the GST tag during protein purification.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 673
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 85
2020 SZPT-CHINA
Description
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. 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.
Result
Expression of GST-TP-1M in E. coli BL21
The recombinant strain E. coli BL21 (DE3) with pGEX-4T-2-TP-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-TP-1M produced 36kDa protein matched well with our GST-TP-1M fusion protein.In contrast, Non-induced E. coli BL21 cells did not express GST or GST-TP-1M.Western blot results showed 36kDa fusion protein carry GST tag. The results showed that the constructed tandem TP-1M 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-TP1M was obtained. The SDS-PAGE electrophoresis detection result is shown in the Fig.4.
| None |