Cloning and Expression of Rat Brain Acetylcholinesterase Enzyme in Escherichia coli

Yıl 2022, Cilt: 12 Sayı: 1, 287 - 296, 01.03.2022

Ahmet Karakuş , Hamid Ceylan , Orhan Erdoğan

https://doi.org/10.21597/jist.962268

Öz

In this study, the gene region of rat (Rattus norvegicus) brain acetylcholinesterase enzyme was amplified in PCR with designed forward and reverse primers and it was ligated into a pET-SUMO vector under suitable conditions. This recombinant vector was transformed to competent Escherichia coli cells and it was grown in liquid LB medium including kanamycin. Colony PCR was performed from growing colony and PCR products were checked with agarose gel electrophoresis. The correct colonies were grown in a liquid medium for plasmid isolation. After plasmid isolation, these recombinant constructs were used for whether the gene inserts properly with cross-PCR. After determining the accuracy of the plasmid, recombinant vectors were transferred into the E. coli BL21 (DE3) cells to perform protein production. Cells were grown in IPTG induced larger media for hours. Enzyme activity and SDS-PAGE analysis were performed from homogenate for each treatment samples.

Anahtar Kelimeler

Cloning, pET-SUMO vector, E. coli, Rattus norvegicus, Acetylcholinesterase

Kaynakça

• Alaşehirli B, 2005, The Inhibitors Of Cholinesterase (Anticholinesterases). Journal of Internal Medical Sciences Pharmacology, 1 (18): 47.
• Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.
• Boggio R, Colombo R, Hay RT, Draetta GF, Chiocca S, 2004. A mechanism for inhibiting the SUMO pathway. Molecular Cell, 16 (4): 549-561.
• Catanzariti AM, Soboleva TA, Jans DA, Board PG, Baker RT, 2004. An efficient system for high‐level expression and easy purification of authentic recombinant proteins. Protein Science, 13 (5): 1331-1339.
• Colovic MB, Krstic DZ, Lazarevic-Pasti TD, Bondzic AM, Vasic VM, 2013. Acetylcholinesterase inhibitors: pharmacology and toxicology. Current Neuropharmacology, 11 (3): 315-335.
• Coussen F, Bonnerot C, Massoulie J, 1995. Stable expression of acetylcholinesterase and associated collagenic subunits in transfected RBL cell lines: production of GPI-anchored dimers and collagen-tailed forms. European Journal of Cell Biology, 67 (3): 254-260.
• De Marco V, Stier G, Blandin S, De Marco A, 2004. The solubility and stability of recombinant proteins are increased by their fusion to NusA. Biochemical and Biophysical Research Communications, 322 (3): 766-771.
• Dorronsoro I, Castro A, Martinez, A, 2003. Peripheral and dual binding site inhibitors of acetylcholinesterase as neurodegenerative disease modifying agents. Expert Opinion on Therapeutic Patents, 13 (11): 1725-1732.
• Ellman GL, Courtney KD, Andres Jr V, Featherstone RM, 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7 (2): 88-95.
• Golde TE, 2003. Alzheimer disease therapy: can the amyloid cascade be halted? The Journal of Clinical Investigation, 111 (1): 11-18.
• Greig NH, Lahiri DK, Sambamurti K, 2002. Butyrylcholinesterase: an important new target in Alzheimer's disease therapy. International Psychogeriatrics, 14 (1): 77-91.
• He G, Sun Y, Li F, 2012. RNA interference of two acetylcholinesterase genes in Plutella xylostella reveals their different functions. Archives of Insect Biochemistry and Physiology, 79 (2): 75-86.
• Holzgrabe U, Kapkova P Alptuzun, V, Scheiber J, Kugelmann E, 2007. Targeting acetylcholinesterase to treat neurodegeneration. Expert Opinion on Therapeutic Targets, 11 (2): 161-179.
• Kim AR, Rylett RJ, Shilton BH. Substrate binding and catalytic mechanism of human choline acetyltransferase. Biochemistry. 2006 Dec 12;45(49):14621-31.Kronman C, Velan B, Gozes Y, Leitner M, Flashner Y, Lazar A, Marcus D, Sery T, Papier Y, Grosfeld H, 1992. Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit. Gene, 121 (2): 295-304.
• LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM, 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology, 11: 187-193.
• Lazar A, Reuveny S, Kronman C, Velan B, Shafferman A, 1993. Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells. Cytotechnolog, 13 (2): 115-123.
• Legay C, Bon S, Vernier P, Coussen F, Massoulie J, 1993. Cloning and expression of a rat acetylcholinesterase subunit: generation of multiple molecular forms and complementarity with a Torpedo collagenic subunit. Journal of Neurochemistry, 60 (1): 337-346.
• Lester HA, 1977. The response to acetylcholine. Scientific American, 236 (2): 106-120.
• Lu W, Jiang J, Yang G, Lai J, Lu K, 2012. Endovascular treatment for the spontaneous rupture of a non-aneurysmal subdiaphragmatic aorta. Vasa, 41 (4): 288-291.
• Lu H, Liu X, Deng Y, Qing H, 2013. DNA methylation, a hand behind neurodegenerative diseases. Frontiers in Aging Neuroscience, 5: 85.
• Massoulie J, Pezzementi L, Bon S, Krejci E, Vallette FM, 1993. Molecular and cellular biology of cholinesterases. Progress in Neurobiology, 41 (1): 31-91.
• McGleenon B, Dynan K, Passmore A, 1999. Acetylcholinesterase inhibitors in Alzheimer’s disease. British Journal of Clinical Pharmacology, 48 (4): 471.
• Méndez V, Avelar E, Morales A, Cervantes M, Araiza A, González D, 2011. A rapid protocol for purification of total RNA for tissues collected from pigs at a slaughterhouse. Genetics and Molecular Research: GMR, 10 (4): 3251-3255.
• Mesulam MM, Guillozet A, Shaw P, Levey A, Duysen E, Lockridge O, 2002. Acetylcholinesterase knockouts establish central cholinergic pathways and can use butyrylcholinesterase to hydrolyze acetylcholine. Neuroscience, 110 (4): 627-639.
• Nachmansohhn, D., 1952. In Modern Trens in Physicology, Biochemistry. Academic Press, New York, 229.
• Nirogi R, Mudigonda K, Kandikere V, Ponnamaneni R, 2010. Quantification of acetylcholine, an essential neurotransmitter, in brain microdialysis samples by liquid chromatography mass spectrometry. Biomedical Chromatography, 24 (1): 39-48.
• Park SE, Kim ND, Yoo YH, 2004. Acetylcholinesterase plays a pivotal role in apoptosome formation. Cancer Research, 64 (24): 9230.
• Picanco-Castro V, Biaggio RT, Cova DT, Swiech K, 2013. Production of recombinant therapeutic proteins in human cells: current achievements and future perspectives. Protein & Peptide Letters, 20 (12): 1373-1381.
• Quinn DM, 1987. Acetylcholinesterase: enzyme structure, reaction dynamics, and virtual transition states. Chemical Reviews, 87 (5): 955-979.
• Snowdon DA, Greiner LH, Mortimer JA, Riley KP, Greiner PA, Markesbery WR, 1997. Brain infarction and the clinical expression of Alzheimer disease. The Nun Study. Jama, 277 (10): 813-817.
• Soreq H, and Seidman S, 2001. Acetylcholinesterase new roles for an old actor. Nature Reviews Neuroscience, 2 (4): 294-302.
• Thomas JG, Baneyx F, 1997. Divergent Effects of Chaperone Overexpression and Ethanol Supplementation on Inclusion Body Formation in Recombinant Escherichia coli. Protein Expression and Purification, 11 (3): 289-296.
• Velan B, Grosfeld H, Kronman C, Leitner M, Gozes Y, Lazar A, Flashner Y, Marcus D, Cohen S, Shafferman A, 1991. The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580-Ala mutant. Journal of Biological Chemistry, 266 (35): 23977-23984.
• Vellom DC, Radic Z, Li Y, Pickering NA, Camp S, Taylor, P, 1993. Amino acid residues controlling acetylcholinesterase and butyrylcholinesterase specificity. Biochemistry, 32 (1): 12-17.
• Wang Z, Li H, Guan W, Ling H, Wang Z, Mu T, Shuler FD, Fang X, 2010. Human SUMO fusion systems enhance protein expression and solubility. Protein Expression and Purification, 73 (2): 203-208.
• Whittaker VP, 1990. The contribution of drugs and toxins to understanding of cholinergic function. Trends in Pharmacological Sciences, 11 (1): 8-13.
• Yamamoto K, Oguri S, Chiba S, Momonoki YS, 2009. Molecular cloning of acetylcholinesterase gene from Salicornia europaea L. Plant Signaling and Behavior, 4 (5): 361-366.
• Ye X, Zhang C, Chen Y, Zhou T, 2015. Upregulation of Acetylcholinesterase Mediated by p53 Contributes to Cisplatin-Induced Apoptosis in Human Breast Cancer Cell. Journal of Cancer, 6 (1): 48-53.
• Zhang Y. Olsen DR, Nguyen KB, Olson PS, Rhodes ET, Mascarenhas D, 1998. Expression of eukaryotic proteins in soluble form in Escherichia coli. Protein Expression and Purification, 12 (2): 159-165.
• Zhang XJ, and Greenberg DS, 2012. Acetylcholinesterase involvement in apoptosis. Frontiers in Molecular Neuroscience,10 (5): 40.