Part:BBa_J18918:Experience
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how you used this part and how it worked out.
Applications of BBa_J18918
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Characterization of BBa_J18918 by 2022 TRIS_iGEM team
Usage
For our project, we aimed to express an antimicrobial peptide (AMP), a modified version of bombolitin BBa_J18918, in E. coli. However, due to its antimicrobial properties, we could not express the modified bombolitin directly.
The mechanism of action of the modified bombolitin lies in its positive charge and hydrophobicity, similar to how other AMPs act. Firstly, positively charged AMPs are attracted to the outer membrane protein of bacterial cells, since the majority of proteins embedded into the outer membrane of bacterial cells are negatively charged, thereby attracting the AMP’s negative charges. This causes the permeability of the membrane to be disrupted, effectively rupturing the membrane and killing the bacteria through lysis. Secondly, hydrophobic AMPs allow it to traverse the membrane and eventually get into the cytoplasm. Once in the cytoplasm, the AMP is able to disrupt a variety of intracellular functions, leading to eventual cell death. Our modified bombolitin is both hydrophobic and cationic, allowing it to act intracellularly and on the membrane. Thus, it is extremely lethal to bacteria, including our expression host.
To overcome this, fusion tags or stabilization tags can be added to the bombolitin in order to conceal its antimicrobial nature by reducing the hydrophobicity and the positive charge. For our project specifically, we used Thioredoxin. We also added a His-tag to allow us to separate the expressed bombolitin from other proteins of the expression host. Once expressed, we must separate the bombolitin sequence from the fusion tag. To do this, we added a TEV cleavage site between the His-tag and the bombolitin sequence, as shown below.
Using a TEV protease, we were able to separate the bombolitin sequence from the rest of the expressed sequence, allowing us to use E. coli to produce our modified bombolitin. Overall, we had very little trouble utilizing the TEV cleavage site and the TEV protease.
Literature review of BBa_J18918 by 2022 TRIS_iGEM team
Team Thailand_RIS (iGEM 2022) used the TEV cleavage site as a crucial part of our project, allowing us to express our modified bombolitin in E. coli, despite its antimicrobial nature. Since TEV protease is widely used for cleaving fused proteins, we decided to do a literature review to add additional information to the registry page of this part. We hope that this will benefit future iGEM teams by allowing them to readily access important information regarding TEV protease and its cleavage site, as many future teams will likely use it as a vital part in their project.
Cleavage Site:
The TEV protease recognizes the linear amino acid sequence E-X-X-Y-X-Q-(G/S/A). The cleaving is always performed between Q and G/S/A. The most efficient substrate for TEV protease is the cleavage site with amino acid sequence as follows: ENLYFQS. Substitutions in positions P2, P4, and P5 still allow cleavage by the TEV protease. However, even relatively conservative substitutions, such as Leu to Phe to P4, results in considerably lower efficiencies. On the other hand, G, S, and A can be interchanged without significant decrease in processing efficiency.
Standard Conditions for Cleaving: Standard reaction buffer:
- 50 mM Tris-HCl (pH 8.0) - 0.5 mM EDTA - 1mM DTT
TEV cleavage reactions are typically left overnight. However, the majority of the cleavage happens in the first few hours and there is no solid evidence to suggest that a prolonged incubation time leads to a proportional increase in cleavage. The optimal temperature is 30-34 ºC, though it is relatively active until as low as 4 °C. Hence, most TEV cleavage reactions are performed at room temperature.
Cleaving Inhibitors:
Because TEV protease is not part of the serine protease family, the following common protein inhibitors will not inhibit its cleaving: PMSF and AEBSF (1mM), TLCK (1mM), Bestatin (1mg/ml), pepstatin A (1mM), EDTA (1mM), and E-64 (3mg/ml). At concentrations of 5mM or above, Zinc ions will inhibit cleaving. Any reagents that react with cysteine will effectively inhibit the activity of TEV protease at the cleaving site. For instance, iodoacetamide or NEM.
References
Fox, J. D. and Waugh, D. S. (2003). Maltose-binding protein as a solubility enhancer. Methods Mol. Biol. 205: 99-117.
Kapust, R. B. and Waugh, D. S. (1999). Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8:1668-1674.
Kapust, R. B. and Waugh, D. S. (2000). Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr. Purif. 19: 312-318.
Kapust, R. B., Tözsér, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Prot. Eng. 14: 993-1000.
Kapust, R. B., Tözsér, J., Copeland, T. D., and Waugh, D. S. (2002a). The P1' specificity of tobacco etch virus protease. Biochem. Biophys. Res. Commun. 294: 949- 955.
Kapust, R. B., Routzahn, K. M., and Waugh, D. S. (2002b). Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant TEV protease in Escherichia coli BL21(DE3). Prot. Expr. Purif. 24: 61-70.
Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30: 544-550.
Mohanty, A. K., Simmons, C.R., and Wiener, M.C. (2003). Inhibition of tobacco etch virus protease activity by detergents. Protein Expr. Purif. 27: 109-114.
Phan, J., Zdanov, A., Evdokimov, A. G., Tropea, J. E., Peters, H. P. K., Kapust, R. B., Li, M., Wlodawer, A., and Waugh, D. S. (2002). Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277: 50564-50572.
Zhang, QY., Yan, ZB., Meng, YM. et al. (2021). Antimicrobial peptides: mechanism of action, activity and clinical potential. Military. Med. Res. 8: 48
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