Coding

Part:BBa_K3487001

Designed by: Jianhua Zhou   Group: iGEM20_SZPT-CHINA   (2020-10-08)
Revision as of 01:44, 21 October 2020 by Linruixin (Talk | contribs) (Construction and Identification of Recombinant Expression Vector of antimicrobial peptides)

TP-ⅠM,A polymer of Tachyplesin Ⅰ

Descrption

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-1M containing 73 amino acids was joined by E, cleavage sites of Glu-C.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



2020 SZPT-CHINA

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

Since TP-Ⅰ is composed of only a few amino acids, it is difficult to biosynthesize directly. Firstly, antimicrobial Peptides Multimer was constructed. TP-ⅠM containing 73 amino acids was joined by E, cleavage sites of Glu-C. The amino acid sequence of TP-ⅠM is shown in Fig.1.

T--SZPT-CHINA--TP-1M 3.png

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

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.

T--SZPT-CHINA--TP-1M 4.png

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.

T--SZPT-CHINA--TP-1M 5.png

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-TP1M produced 36kDa protein matched well with our GST-TP1M 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.

T--SZPT-CHINA--TP-1M 6.png

Expression of prepared antimicrobial peptide activity results

After the purified fusion protein is digested by thrombin and glutamate endopeptidase, antimicrobial peptide monomers are obtained. The antibacterial results are shown in the Fig.5.

T--SZPT-CHINA--TP-1M 7.png

The purified TP-1 has a good inhibitory effect on S.mutans.

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