Difference between revisions of "Part:BBa K3487001"
| Line 15: | Line 15: | ||
| − | + | =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. | ||
| + | |||
| + | [[File:T--SZPT-CHINA--TP-1M 3.png|center|500px|]] | ||
| + | |||
| + | ==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|]] | ||
| + | |||
| + | ===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=== | ===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
- 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-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.
