Pyruvate, phosphate dikinase (PPDK)
PPDK was used as a biomarker for the detection of amebiasis - a disease caused by Entamoeba histolytica. This was achieved through a method called SELEX which uses in vitro directed evolution to find a suitable single stranded DNA detection probe called an aptamer.
Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.
The enzyme was found and mostly studied in plants where it catalyzes the reaction of pyruvate phosphorylation using ATP although it is also found in some bacteria and our target organism – entamoeba histolytica as well. It’s a part of the transferase family of enzymes, more specifically phosphotransferases. The reaction is vital in photosynthesis and gluconeogenesis and is the opposite of the glycolysis reaction facilitated by pyruvate kinase.
Usage in our project
The specific role of this protein in our target organism was not that important to us because we only needed the protein to be excreted to the amoeba’s environment for detection. This protein was a perfect fit for us because it has shown some promise as a biomarker for the ameba in previous sources[1, 2] and it is not toxic to the E. coli cell so we can make enough of the recombinant protein to develop our test. Besides the actual SELEX process in a lab we also modeled our protein so we could dock different aptamers to it in silico.
Fig. 1 A 3D model of pyruvate, phosphate dikinase (PPDK) (UniProtKB - P37213)
Recombinant expression and purification
We were not able to purify the soluble fraction of the protein, but we managed to work around that by stabilizing the denatured recombinant protein in a buffer suitable for aptamers. At first we found out that PPDK is solubilized at 6M concentration of urea, and then, after dialyzing it against a bunch of different lysis buffer additives[3, 4] found out that it is successfully stabilized in a buffer containing arginine of a 0.375M concentration.
Fig. 2 An SDS gel showing the PPDK solubilization at 6M urea concentration
Fig. 3 An SDS gel showing the stabilization effect of arginine
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal XhoI site found at 2656
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1149
Illegal NgoMIV site found at 2409
Illegal AgeI site found at 2187
- 1000Illegal BsaI.rc site found at 1285
Illegal SapI.rc site found at 1753
- Wong, W. K., Tan, Z. N., Othman, N., Lim, B. H., Mohamed, Z., Olivos Garcia, A., & Noordin, R. (2011). Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker. Clinical and Vaccine Immunology, 18(11), 1913–1917. https://journals.asm.org/doi/full/10.1128/CVI.05356-11
- Saidin, S., Yunus, H. M., Zakaria, D. N., Razak, A. K., Huat, B. L., Othman, N., & Noordin, R. (2014). Erratum to: Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess [BMC Infect Dis, 14, (2014), 182, doi: 10.1186/1471-2334-14-182]. BMC Infectious Diseases, 14(1), 1–9. https://doi.org/10.1186/1471-2334-14-533
- Leibly, D. J., Nguyen, T. N., Kao, L. T., Hewitt, S. N., Barrett, L. K., & Van Voorhis, W. C. (2012). Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins. PLoS ONE, 7(12), e52482. https://www.doi.org/10.1371/journal.pone.0052482
- Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J., & Arakawa, T. (2004). Role of Arginine in Protein Refolding, Solubilization, and Purification. Biotechnology Progress, 20(5), 1301–1308. https://doi.org/10.1021/bp0498793