Revision as of 02:33, 22 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 673
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE 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

Construction and Identification of Recombinant Expression Vector of antimicrobial peptides

We cloned the TP-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 TP-ⅠM, shown in Fig.2. Hence, the recombinant expression vector pGEX-4T-2-TP-ⅠM was successfully constructed.

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.

@@ Line 18: / Line 18: @@
 <!-- -->
-==2020 SZPT-CHINA==
+=2020 SZPT-CHINA=
-===Description===
+==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===
+==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.
-[[File:T--SZPT-CHINA--GST-TP-1M.png|center|500px|SDS-PAGE analysis of expression and purification of GST-TP1M in E. coli BL21
+===Construction and Identification of Recombinant Expression Vector of antimicrobial peptides===
-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
+We cloned the TP-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 TP-ⅠM, shown in Fig.2. Hence, the recombinant expression vector pGEX-4T-2-TP-ⅠM was successfully constructed.
-]]
+[[File:T--SZPT-CHINA--TP-1M 4.png|center|700px|]]
+===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.
+[[File:T--SZPT-CHINA--TP-1M 5.png|center|500px]]
+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.
+[[File:T--SZPT-CHINA--TP-1M 6.png|center|800px|]]