Difference between revisions of "Part:BBa K3487001"
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<partinfo>BBa_K3487001 short</partinfo>
 
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===Descrption===
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===Description===
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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.
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A long-chain protein formed by polymerizing four identical short TP-I monomer peptides.
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===Usage and Biology===
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After the tp1 polymer is expressed, it is inactive. It will be expressed as a long-chain protein by the engineered bacteria and taken to the extracellular environment by the signal peptide and then hydrolyzed into four active short peptides.
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[[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.]]
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===Usage and Biology===
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===Characterization===
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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.
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===Sequence and Features===
 
===Sequence and Features===
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<partinfo>BBa_K3487001 parameters</partinfo>
 
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=2020 SZPT-CHINA=
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==Construction of the antimicrobial Peptides Multimer(TP-ⅠM)==
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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.
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[[File:T--SZPT-CHINA--TP-1M 3.png|center|500px|]]
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==Fusion expression of TP-ⅠM in <i>E. coli</i> system==
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===Construction and Identification of Recombinant Expression Vector of antimicrobial peptides===
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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.
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[[File:T--SZPT-CHINA--TP-1M 4.png|center|700px|]]
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===Expression of GST-TP-1M in <i>E. coli</i> BL21===
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The recombinant strain <i>E. coli</i> 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.
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[[File:T--SZPT-CHINA--TP-1M 5.png|center|500px]]
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After IPTG induction, the <i>E. coli</i> BL21 transferred with pGEX-4T-2 vector produced 26kDa GST protein and <i>E. coli</i> BL21 transferred with pGEX-4T-2-TP-1M produced 36kDa protein matched well with our GST-TP-1M fusion protein.In contrast, Non-induced <i>E. coli</i> 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.
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===Fusion protein purification results===
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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.
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[[File:T--SZPT-CHINA--TP-1M 6.png|center|800px|]]
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===Expression of prepared antimicrobial peptide activity results===
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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.
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[[File:T--SZPT-CHINA--TP-1M 7.png|center|500px]]
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The purified TP-1 has a good inhibitory effect on <i>S.mutans</i>.
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==Expression of TP-ⅠM in lactic acid bacteria==
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===Construction of Recombinant Plasmid pMG36N-TP-ⅠM===
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The oral cavity is in an acidic environment when the human body suffers from mild dental caries. Therefore, the acid-inducible promoter P170 was used for TP-ⅠM expression and secretion signal peptide SPusp45 were used to secrete the target protein directly.The recombinant vector named pMG36N-TP-ⅠM and was confirmed by PCR analysis. As shown in Fig.6.
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[[File:T--SZPT-CHINA--TP-1M 8.png|center|800px]]
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The fragment size is the same as TP-ⅠM. The results showed that the recombinant expression vector pMG36N-TP-ⅠM was successfully constructed.
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===Reference===
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[1]Zhou L, Liu Z, Xu G, et al. Expression of Melittin in Fusion with GST in Escherichia coli and Its Purification as a Pure Peptide with Good Bacteriostatic Efficacy[J]. ACS omega, 2020, 5(16): 9251-9258.
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[2]Jin Yuanbao,Wang Ying,Liu Liping,Wang Qian,Jin Gang,Zhang Lijun,Dai Jianguo.Study on the effect of Limulus I on Proteus vulgaris[J].Biotechnology Bulletin,2014(05):190-196.

Latest revision as of 03:06, 28 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.

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-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.

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.

Expression of TP-ⅠM in lactic acid bacteria

Construction of Recombinant Plasmid pMG36N-TP-ⅠM

The oral cavity is in an acidic environment when the human body suffers from mild dental caries. Therefore, the acid-inducible promoter P170 was used for TP-ⅠM expression and secretion signal peptide SPusp45 were used to secrete the target protein directly.The recombinant vector named pMG36N-TP-ⅠM and was confirmed by PCR analysis. As shown in Fig.6.

T--SZPT-CHINA--TP-1M 8.png

The fragment size is the same as TP-ⅠM. The results showed that the recombinant expression vector pMG36N-TP-ⅠM was successfully constructed.

Reference

[1]Zhou L, Liu Z, Xu G, et al. Expression of Melittin in Fusion with GST in Escherichia coli and Its Purification as a Pure Peptide with Good Bacteriostatic Efficacy[J]. ACS omega, 2020, 5(16): 9251-9258.

[2]Jin Yuanbao,Wang Ying,Liu Liping,Wang Qian,Jin Gang,Zhang Lijun,Dai Jianguo.Study on the effect of Limulus I on Proteus vulgaris[J].Biotechnology Bulletin,2014(05):190-196.