To test our hypothesis, we first designed the original DNA sequence to be a 6xHis-tag at the 5’ end with a single peptide sequence directly attached to at the 3’ end. The DNA sequence was then cloned onto plasmid pET-30a (+) downstream of a T7 promoter. The plasmid was first amplified in the DH5α strain of E. coli and uptaken by the competent BL21(DE3) strain of E. coli which expresses T7 RNA polymerase allowing rapid expression of the peptide. The expression of the peptide was induced at 37 degrees Celcius and 20 degrees Celcius with IPTG of 0.5 mM right at the exponential growth stage of E. coli. No significant expression was detected in either common SDS-page gel electrophoresis or tricine-SDS-page gel electrophoresis for small peptides. Recognizable cell precipitation can be observed after the IPTG induction or even after 12 hours of incubation without IPTG, implying that the peptide cannot be directly synthesized either because of its extremely small size (2 kDa) or because of its strong toxicity towards bacterial cells.
The DNA sequence clearly required further modification. To shield the toxicity of the peptide, a SUMO-tag with a linker sequence of GGGGS at its 5’ end is added to the 3’end of the 6x His-tag, which can be cut away altogether by SUMO protease in later processes. To increase the algicidal activity, following the “His-tag, linker sequence, SUMO-tag” sequence, a triple-tandem peptide sequence with GGGGS between each complete peptide is added to its 3’ end. The linker sequence (GGGGS) maintains the flexibility of rotation or movement of every single peptide sequence, allowing the tight contact of each peptide sequence to the surface of algae without steric hindrance (Chen et al., 2013). This plasmid is named SUMO-H3, while another SUMO-H1 plasmid with only one single peptide sequence is built up to examine the potentially increased algicidal effect of the triple-tandem peptide.
Figure 3. Design of the SUMO-His-Tag-Tandem Plasmid
+
<br><br>
+
<h3> Result </h3>
+
By using SUMO tag to shield the toxicity of the algicidal peptide to the engineering bacteria, we successfully synthesized and purified the peptide. Compared with non-modified peptides, the yield increased significantly.
+
<br><br>
Revision as of 02:34, 22 October 2021
Tandem Algicidal Peptide SUMO His Tag Expression
Algicidal peptide coding gene after the codon optimization. The products are algicidal peptides that can be used as a highly efficient and targeted measure to terminate urgent ongoing cyanoHAB. The synthesized fragment contains (1) His-tag at the beginning for future purification, (2) sumo-tag for masking the toxicity to the host and providing cleavage site to remove the tags attached to the peptide, (3) triple peptide tandem with the GGGGS linkers. We used the T7 promoter to control the peptide expression. The ribosomal binding site and T7 terminator were added for full protein expression.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 545
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 319
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]
Design-first trial
To test our hypothesis, we first designed the original DNA sequence to be a 6xHis-tag at the 5’ end with a single peptide sequence directly attached to at the 3’ end. The DNA sequence was then cloned onto plasmid pET-30a (+) downstream of a T7 promoter. The plasmid was first amplified in the DH5α strain of E. coli and uptaken by the competent BL21(DE3) strain of E. coli which expresses T7 RNA polymerase allowing rapid expression of the peptide. The expression of the peptide was induced at 37 degrees Celcius and 20 degrees Celcius with IPTG of 0.5 mM right at the exponential growth stage of E. coli. No significant expression was detected in either common SDS-page gel electrophoresis or tricine-SDS-page gel electrophoresis for small peptides. Recognizable cell precipitation can be observed after the IPTG induction or even after 12 hours of incubation without IPTG, implying that the peptide cannot be directly synthesized either because of its extremely small size (2 kDa) or because of its strong toxicity towards bacterial cells.
The DNA sequence clearly required further modification. To shield the toxicity of the peptide, a SUMO-tag with a linker sequence of GGGGS at its 5’ end is added to the 3’end of the 6x His-tag, which can be cut away altogether by SUMO protease in later processes. To increase the algicidal activity, following the “His-tag, linker sequence, SUMO-tag” sequence, a triple-tandem peptide sequence with GGGGS between each complete peptide is added to its 3’ end. The linker sequence (GGGGS) maintains the flexibility of rotation or movement of every single peptide sequence, allowing the tight contact of each peptide sequence to the surface of algae without steric hindrance (Chen et al., 2013). This plasmid is named SUMO-H3, while another SUMO-H1 plasmid with only one single peptide sequence is built up to examine the potentially increased algicidal effect of the triple-tandem peptide.
Figure 3. Design of the SUMO-His-Tag-Tandem Plasmid
Result
By using SUMO tag to shield the toxicity of the algicidal peptide to the engineering bacteria, we successfully synthesized and purified the peptide. Compared with non-modified peptides, the yield increased significantly.
" alt="Image" class="img-fluid">
</a >
" alt="Image" class="img-fluid">
</a >
" alt="Image" class="img-fluid">
</a >