Difference between revisions of "Part:BBa K3487013"
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<partinfo>BBa_K3487013 parameters</partinfo> | <partinfo>BBa_K3487013 parameters</partinfo> | ||
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| + | ==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=== | ||
| + | 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. | ||
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| + | [[File:T--SZPT-CHINA--GST-TP-1M.png|center|500px|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 | ||
| + | ]] | ||
Revision as of 09:02, 19 October 2020
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
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.
