Difference between revisions of "Part:BBa K3487001"

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==2020 SZPT-CHINA==
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=2020 SZPT-CHINA=
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==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.
 +
 
 +
[[File:T--SZPT-CHINA--TP-1M 3.png|center|500px|]]
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==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.
 +
 
 +
[[File:T--SZPT-CHINA--TP-1M 4.png|center|500px|]]
 +
 
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===Expression of GST-TP-1M in E. coli BL21===
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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-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.
 +
 
 +
 
 
===Characterization===
 
===Characterization===
 
By externally synthesizing an expression vector with Tachyplesin I multimers, transferring the vector into BL21 for antibiotic screening to obtain a single clone, culture it in liquid medium to OD600 and add IPTG to induce 12 hours to obtain recombinant protein, and collect After ultrafiltration and concentration, purified Tachyplesin I polymer was obtained. SDS-PAGE electrophoresis identification showed that the target protein had been obtained.
 
By externally synthesizing an expression vector with Tachyplesin I multimers, transferring the vector into BL21 for antibiotic screening to obtain a single clone, culture it in liquid medium to OD600 and add IPTG to induce 12 hours to obtain recombinant protein, and collect After ultrafiltration and concentration, purified Tachyplesin I polymer was obtained. SDS-PAGE electrophoresis identification showed that the target protein had been obtained.
 
[[File:T--SZPT-CHINA--TP-1M.png|400px|thumb|center|By externally synthesizing an expression vector with Tachyplesin I multimers, transferring the vector into BL21 for antibiotic screening to obtain a single clone, culture it in liquid medium to OD600 and add IPTG to induce 12 hours to obtain recombinant protein, and collect After ultrafiltration and concentration, purified Tachyplesin I polymer was obtained. SDS-PAGE electrophoresis identification showed that the target protein had been obtained.]]
 
[[File:T--SZPT-CHINA--TP-1M.png|400px|thumb|center|By externally synthesizing an expression vector with Tachyplesin I multimers, transferring the vector into BL21 for antibiotic screening to obtain a single clone, culture it in liquid medium to OD600 and add IPTG to induce 12 hours to obtain recombinant protein, and collect After ultrafiltration and concentration, purified Tachyplesin I polymer was obtained. SDS-PAGE electrophoresis identification showed that the target protein had been obtained.]]

Revision as of 12:35, 20 October 2020

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. 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.


Characterization

By externally synthesizing an expression vector with Tachyplesin I multimers, transferring the vector into BL21 for antibiotic screening to obtain a single clone, culture it in liquid medium to OD600 and add IPTG to induce 12 hours to obtain recombinant protein, and collect After ultrafiltration and concentration, purified Tachyplesin I polymer was obtained. SDS-PAGE electrophoresis identification showed that the target protein had been obtained.

By externally synthesizing an expression vector with Tachyplesin I multimers, transferring the vector into BL21 for antibiotic screening to obtain a single clone, culture it in liquid medium to OD600 and add IPTG to induce 12 hours to obtain recombinant protein, and collect After ultrafiltration and concentration, purified Tachyplesin I polymer was obtained. SDS-PAGE electrophoresis identification showed that the target protein had been obtained.