Difference between revisions of "Part:BBa K1835501"
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The obtained construct was transformed into E. coli T7 Express competent cells using heat shock protocol, and plated on agar plates containing 100 µg/mL ampicillin. Colonies were checked for positive clones using tachyplesin forward and T7 reverse primers. | The obtained construct was transformed into E. coli T7 Express competent cells using heat shock protocol, and plated on agar plates containing 100 µg/mL ampicillin. Colonies were checked for positive clones using tachyplesin forward and T7 reverse primers. | ||
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====Agarose gel of tachyplesin gene after amplification (a) and after positive clone check (b)==== | ====Agarose gel of tachyplesin gene after amplification (a) and after positive clone check (b)==== | ||
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We tried to purify the GB1-tachyplesin gene using Ni-NTA gravity flow chromatography with two different methods. For the first method, we lysed cells with sonication in mild conditions (50 mM Tris pH 8, 300 mM NaCl, 25 mM imidazole). Same buffer was used for chromatography, after lysis protein was centrifuged and supernatant applied to a previously equilibrated column. Protein was eluted with 50 mM Tris pH 8, 300 mM NaCl, 250 mM imidazole buffer. In second method, we lysed cells in denaturing conditions (8 M urea, 50 mM Tris pH 8, 300 mM NaCl, 25 mM imidazole), centrifuged, applied supernatant to column and eluted with 8 M urea, 50 mM Tris pH 8, 300 mM NaCl, 250 mM imidazole buffer. After SDS-PAGE analysis, we saw that despite the fact that the GB1 solubility tag attached to the peptide, it was almost insoluble in mild lysis conditions as almost all of the protein (MW = 10.8 kDa) was found in the cell pellet. Ni-NTA purification in denaturing conditions yielded much more protein in eluted fraction. | We tried to purify the GB1-tachyplesin gene using Ni-NTA gravity flow chromatography with two different methods. For the first method, we lysed cells with sonication in mild conditions (50 mM Tris pH 8, 300 mM NaCl, 25 mM imidazole). Same buffer was used for chromatography, after lysis protein was centrifuged and supernatant applied to a previously equilibrated column. Protein was eluted with 50 mM Tris pH 8, 300 mM NaCl, 250 mM imidazole buffer. In second method, we lysed cells in denaturing conditions (8 M urea, 50 mM Tris pH 8, 300 mM NaCl, 25 mM imidazole), centrifuged, applied supernatant to column and eluted with 8 M urea, 50 mM Tris pH 8, 300 mM NaCl, 250 mM imidazole buffer. After SDS-PAGE analysis, we saw that despite the fact that the GB1 solubility tag attached to the peptide, it was almost insoluble in mild lysis conditions as almost all of the protein (MW = 10.8 kDa) was found in the cell pellet. Ni-NTA purification in denaturing conditions yielded much more protein in eluted fraction. | ||
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====SDS-PAGE analysis of tachyplesin I purification with or without urea==== | ====SDS-PAGE analysis of tachyplesin I purification with or without urea==== | ||
1: cell pellet after lysis in buffer without urea | 1: cell pellet after lysis in buffer without urea | ||
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Eluted protein in 8 M urea was diluted to 25 mL in lysis buffer and dialysed against 20 mM Tris pH 8, 200 mM NaCl, 2 mM DTT buffer at 4 ⁰C overnight. Unfortunately, there was a lot of precipitation in the dialysis membrane the next morning. We removed precipitation via centrifugation, concentrated the supernatant to 3 mL and applied it to HiLoad 16/600 75 pg SEC column. Unfortunately, no protein peaks could be seen in this chromatography, therefore we concluded that all protein had been precipitated. We repeated expression and Ni-NTA purification in urea one more time, this time we dialysed the protein against 50 mM Tris, 500 mM NaCl, 5 mM DTT buffer at 4 ⁰C overnight. There was still a lot of precipitation. When we applied supernatant to HiLoad 16/600 75 pg SEC column, we saw that protein was eluted right after the void volume of the column (45 mL), thus leading to the conclusion that all protein was in aggregates. Therefore we can say that GB1 tag can yield impressive protein yields in the expression process but unfortunately the purification process is still very complicated. | Eluted protein in 8 M urea was diluted to 25 mL in lysis buffer and dialysed against 20 mM Tris pH 8, 200 mM NaCl, 2 mM DTT buffer at 4 ⁰C overnight. Unfortunately, there was a lot of precipitation in the dialysis membrane the next morning. We removed precipitation via centrifugation, concentrated the supernatant to 3 mL and applied it to HiLoad 16/600 75 pg SEC column. Unfortunately, no protein peaks could be seen in this chromatography, therefore we concluded that all protein had been precipitated. We repeated expression and Ni-NTA purification in urea one more time, this time we dialysed the protein against 50 mM Tris, 500 mM NaCl, 5 mM DTT buffer at 4 ⁰C overnight. There was still a lot of precipitation. When we applied supernatant to HiLoad 16/600 75 pg SEC column, we saw that protein was eluted right after the void volume of the column (45 mL), thus leading to the conclusion that all protein was in aggregates. Therefore we can say that GB1 tag can yield impressive protein yields in the expression process but unfortunately the purification process is still very complicated. | ||
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====Size exclusion chromatography of GB1-tachyplesin after refolding==== | ====Size exclusion chromatography of GB1-tachyplesin after refolding==== | ||
Latest revision as of 14:46, 12 October 2023
Tachyplesin-I
Tachyplesin I (TP-I) is a cysteine-rich antimicrobial peptide derived from the hemocytes of horseshoe crabs. It is very similar in structure, sequence, and function to Protegrin-1, containing the same disulfide bridges and resulting beta-sheet secondary structure which allows it to form pores in the membrane. This antimicrobial peptide was the first gene that we designed. Similar to how St. Andrews created BBa_K117000, the gene sequence for TP-I was derived by translating the amino acid residues to their most common codon triplet, putting those triplets in order, and adding start and stop codons.
Further research into the action of tachyplesin and protegrin revealed that in addition to having antimicrobial ability, both peptides also exhibited some degree of hemolytic activity against human erythrocytes. In the interest of safety, we wanted to find a way to get rid of the hemolytic activity without abolishing the antimicrobial action. Some more digging led us to research that had been done on versions of both of these antimicrobial peptides in which all cysteine residues had been removed. For tachyplesin, it was found that while the deletion of cysteines somewhat decreased the peptides antibiotic capacity, it completely got rid of its ability to lyse human red blood cells. Likewise, the deletion of cysteines in protegrin was found to have little effect on its ability to kill bacteria, however there was no investigation into its hemolytic activity. Based on this information, we decided to develop genes for these cysteine-deleted antimicrobial peptides and investigate them further.
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-CHIAN
Characterization
As a broad-spectrum antimicrobial peptide, TP-1 has good inhibitory effect on most microorganisms. This year, our project use TP-1 as the antibacterial module of Streptococcus mutans. The following is the results of our Bacteriostatic Experiment. As shown in following, the MIC value of TP-1 against Streptococcus mutans is 8 μg/mL, indicating that TP-1 has a good inhibitory effect on Streptococcus mutans.
2021 HKIS
HKIS Improvement
A AI model machine learning has been ran to improve the cytotoxicity of the peptide and generated several mutant candidates.
2023 Latvia-Riga
Cloning
We cloned tachyplesin I gene in pExp-His-GB1-TEV expression vector using Gibson assembly.
Expression
The obtained construct was transformed into E. coli T7 Express competent cells using heat shock protocol, and plated on agar plates containing 100 µg/mL ampicillin. Colonies were checked for positive clones using tachyplesin forward and T7 reverse primers.
Agarose gel of tachyplesin gene after amplification (a) and after positive clone check (b)
Lanes containing tachyplesin samples labeled in yellow
Tachyplesin I was expressed in E. coli T7 Express cells. A colony was inoculated in 5 mL LB medium containing 100 µg/mL ampicillin and grown overnight at 30 ⁰C with shaking at 200 rpm. The next day overnight culture was added to 500 mL LB medium containing 100 µg/mL ampicillin and grown at 37 ⁰C until OD600 reached 0.6. Then temperature was reduced to 18⁰C, and after 30 minutes protein expression was induced with 0.2 mM IPTG. Expression was carried out for 18 hours, then cells were harvested with centrifugation at 7000 xg.
Purification
We tried to purify the GB1-tachyplesin gene using Ni-NTA gravity flow chromatography with two different methods. For the first method, we lysed cells with sonication in mild conditions (50 mM Tris pH 8, 300 mM NaCl, 25 mM imidazole). Same buffer was used for chromatography, after lysis protein was centrifuged and supernatant applied to a previously equilibrated column. Protein was eluted with 50 mM Tris pH 8, 300 mM NaCl, 250 mM imidazole buffer. In second method, we lysed cells in denaturing conditions (8 M urea, 50 mM Tris pH 8, 300 mM NaCl, 25 mM imidazole), centrifuged, applied supernatant to column and eluted with 8 M urea, 50 mM Tris pH 8, 300 mM NaCl, 250 mM imidazole buffer. After SDS-PAGE analysis, we saw that despite the fact that the GB1 solubility tag attached to the peptide, it was almost insoluble in mild lysis conditions as almost all of the protein (MW = 10.8 kDa) was found in the cell pellet. Ni-NTA purification in denaturing conditions yielded much more protein in eluted fraction.
SDS-PAGE analysis of tachyplesin I purification with or without urea
1: cell pellet after lysis in buffer without urea 2: supernatant after lysis in buffer without urea 3: eluted fraction after Ni-NTA chromatography in buffer without urea 4: supernatant after lysis in buffer with 8 M urea 5: eluted fraction after Ni-NTA chromatography in buffer with urea
Eluted protein in 8 M urea was diluted to 25 mL in lysis buffer and dialysed against 20 mM Tris pH 8, 200 mM NaCl, 2 mM DTT buffer at 4 ⁰C overnight. Unfortunately, there was a lot of precipitation in the dialysis membrane the next morning. We removed precipitation via centrifugation, concentrated the supernatant to 3 mL and applied it to HiLoad 16/600 75 pg SEC column. Unfortunately, no protein peaks could be seen in this chromatography, therefore we concluded that all protein had been precipitated. We repeated expression and Ni-NTA purification in urea one more time, this time we dialysed the protein against 50 mM Tris, 500 mM NaCl, 5 mM DTT buffer at 4 ⁰C overnight. There was still a lot of precipitation. When we applied supernatant to HiLoad 16/600 75 pg SEC column, we saw that protein was eluted right after the void volume of the column (45 mL), thus leading to the conclusion that all protein was in aggregates. Therefore we can say that GB1 tag can yield impressive protein yields in the expression process but unfortunately the purification process is still very complicated.
