Difference between revisions of "Part:BBa K3487001"
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[[File:T--SZPT-CHINA--TP-1M 3.png|center|500px|]] | [[File:T--SZPT-CHINA--TP-1M 3.png|center|500px|]] | ||
| − | ==Fusion expression of TP-ⅠM in E. coli system== | + | ==Fusion expression of TP-ⅠM in <i>E. coli</i> system== |
===Construction and Identification of Recombinant Expression Vector of antimicrobial peptides=== | ===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. | 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| | + | [[File:T--SZPT-CHINA--TP-1M 4.png|center|700px|]] |
| − | ===Expression of GST-TP-1M in E. coli BL21=== | + | ===Expression of GST-TP-1M in <i>E. coli</i> 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 | + | 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]] | [[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. | ||
===Fusion protein purification results=== | ===Fusion protein purification results=== | ||
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[[File:T--SZPT-CHINA--TP-1M 6.png|center|800px|]] | [[File:T--SZPT-CHINA--TP-1M 6.png|center|800px|]] | ||
| − | ==Expression of prepared antimicrobial peptide activity results== | + | ===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. | + | 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. |
| + | |||
| + | [[File:T--SZPT-CHINA--TP-1M 7.png|center|500px]] | ||
| + | |||
| + | The purified TP-1 has a good inhibitory effect on <i>S.mutans</i>. | ||
| + | |||
| + | ==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. | ||
| + | |||
| + | [[File:T--SZPT-CHINA--TP-1M 8.png|center|800px]] | ||
| + | |||
| + | 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. | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
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.
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.
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.
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.