High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

doi:10.1016/S2095-3119(16)61508-3

Journal of Integrative Agriculture ›› 2017, Vol. 16 ›› Issue (06): 1358-1366.DOI: 10.1016/S2095-3119(16)61508-3

收稿日期:2016-06-16 出版日期:2017-06-20 发布日期:2017-06-08

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

LIU Niu¹, CHE Jun^{1, 2}, LAI Duo^{1, 3}, WEN Jin-jun¹, XU Han-hong¹

¹State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources & Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education/South China Agricultural University, Guangzhou 510642, P.R.China
²Shenzhen Longgang Inspection Station of Non-pollution Agricultural Products, Shenzhen 510172, P.R.China
³Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China

Received:2016-06-16 Online:2017-06-20 Published:2017-06-08
Contact: XU Han-hong, Tel: +86-20-85285127, E-mail: hhxu@scau.edu.cn
Supported by:
This work was supported by a grant from the Public Benefit Research Foundation of China (200903052) and the Science and Technology Department of Guangdong Province, China (2009A020101003).

摘要/Abstract

Abstract: The acetylcholinesterase 2 (AChE2) cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115. One transformant with high-level expression of the recombinant AChE (rAChE, 23.2 U mL^–1 in supernatant) was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate. The maximum production of rAChE reached about 11.8 mg of the enzyme protein per liter of culture. The rAChE was ?rst precipitated with ammonium sulfate (50% saturation) and then puri?ed with procainamide af?nity column chromatography. The enzyme was puri?ed 12.1-fold with a yield of 22.8% and a high speci?c activity of 448.3 U mg^–1. It was sensitive to inhibition by methamidophos and pirimicarb, the calculated 50% inhibitory concentration (IC₅₀) values of the two pesticides were 0.357 and 0.888 mg L^–1, respectively, and the calculated 70% inhibitory concentration (IC₇₀) values were 0.521 and 0.839 mg L^–1, respectively. The results suggested that it has a potential application in the detection of pesticide residues.

Key words: Plutella xylostella, acetylcholinesterase, Pichia pastoris,, high-level expression

. [J]. Journal of Integrative Agriculture, 2017, 16(06): 1358-1366.

LIU Niu, CHE Jun, LAI Duo, WEN Jin-jun, XU Han-hong . High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application[J]. Journal of Integrative Agriculture, 2017, 16(06): 1358-1366.

参考文献

Baeka J H, Kima J I, Lee D W, Chung B K, Miyata T, Lee S H. 2005. Identi?cation and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella. Pesticide Biochemistry and Physiology, 81, 164–175.
Benting J, Nauen R. 2004. Biochemical evidence that an S431F mutation in acetylcholinesterase-1 of Aphis gossypii mediates resistance to pirimicarb and omethoate. Pest Management Science, 60, 1051–1055.
Bradford M M. 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.
Chen Z, Newcomb R, Forbes E, McKenzie J, Batterham P. 2001. The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blow?y, Lucilia cuprina. Insect Biochemistry and Molecular Biology, 31, 805–816.
Ellman G L, Courtney K D, Andres V J, Feather-Stone R M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 88–95.
Fournier D, Mutero A. 1994. Modification of acetylcholinesterase as a mechanism of resistance to insecticides. Comparative Biochemistry and Physiology (Part C: Pharmacology, Toxicology & Endocrinology), 108, 19–31.
Golombieski J I, Marchesan E, Camargo E R, Salbego J, Baumart J S, Loro V L, de Oliveira Machado S L, Zanella R, Baldisserotto B. 2008. Acetylcholinesterase enzyme activity in carp brain and muscle after acute exposure to diafuran. Journal of Agricultural Science, 65, 340–345.
Hall L M C, Spierer P. 1986. The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5´ leader. The EMBO Journal, 5, 2949–2954.
Heim J, Shmidt-Dannert C, Atomi H, Schmid R. 1997. Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli. Biochimica et Biophysica Acta, 1396, 306–319.
Ilg T, Schmalz S, Werr M, Cramer J. 2010. Acetylcholinesterases of the cat ?ea Ctenocephalides felis: Identi?cation of two distinct genes and biochemical characterization of recombinant and in vivo enzyme activities. Insect Biochemistry and Molecular Biology, 40, 153–164.
Jiang X, Qu M, Denholm I, Fang J, Jiang W, Han Z. 2009. Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae). Biochemical and Biophysical Research Communications, 378, 269–272.
Khajehali J, Van Leeuwen T, Grispou M, Morou E, Alout H, Weill M, Tirry L, Vontas J, Tsagkarakou A. 2010. Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates. Pest Management Science, 66, 220–228.
Kozaki T, Shono T, Tomita T, Kono Y. 2001. Fenitroxon insensitive acetylcholinesterases of the house?y, Musca domestica associated with point mutations. Insect Biochemistry and Molecular Biology, 31, 991–997.
Lang G J, Shang J Y, Chen Y X, Cui Y J, Wang Q, Tang Z H, Zhang C X. 2010. Expression of the house?y acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues. World Journal of Microbiology and Biotechnology, 26, 1795–1801.
Lee D W, Choi J Y, Kim W T, Je Y H, Song J T, Chung B K, Boo K S, Koh Y H. 2007. Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.). Biochemical and Biophysical Research Communications, 353, 591–597.
Li F, Han Z J. 2004. Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover. Insect Biochemistry and Molecular Biology, 34, 397–405.
Li S, Sing S, Wang Z. 2011. Improved expression of Rhizopus oryzae α-amylase in the methylotrophic yeast Pichia pastoris. Protein Expression and Purification, 79, 142–148.
Lin-Cereghino J, Hashimoto M D, Moy A, Castelo J, Orazem C C, Kuo P, Xiong S, Gandhi V, Hatae C T, Chan A, Lin-Cereghino G P. 2008. Direct selection of Pichia pastoris expression strains using new G418 resistance vectors. Yeast, 25, 293–299.
Lu Y, Pang Y P, Park Y, Gao X, Yao J, Zhang X, Zhu K Y. 2012. Genome organization, phylogenies, expression patterns, and three-dimensional protein models of two acetylcholinesterase genes from the red flour beetle. PLOS ONE, 7, e32288.
Ma X, Tan J, Wei D, Zhu P, Sun M. 2006. High-level secretion and purification of recombinant acetylcholinesterase from human cerebral tissue in P. pastoris and identification by chromogenic reaction. Applied Microbiology and Biotechnology, 72, 316–322.
Marx H, Mecklenbräuker A, Gasser B, Sauer M, Mattanovich D. 2009. Directed gene copy number ampli?cation in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Research, 9, 1260–1270.
Mendelson I, Kronman C, Ariel N, Shafferman A, Velan B. 1998. Bovine acetylcholinesterase: Cloning, expression and characterization. International Journal of Biochemistry, 334, 251–259.
Morel N, Massoulié J. 1997. Expression and processing of vertebrate acetylcholinesterase in the yeast Pichia pastoris. Biochemical Journal, 328, 121–129.
Mutero A, Pralavorio M, Bride J M, Fournier D. 1994. Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase. Proceedings of the National Academy of Sciences of the United States of America, 91, 5922–5926.
Myers E W, Sutton G G, Delcher A L, Dew I M, Fasulo D P, Flanigan M J, Kravitz S A, Mobarry C M, Reinert K H, Remington K A, Anson E L, Bolanos R A, Chou H H, Jordan C M, Halpern A L, Lonardi S, Beasley E M, Brandon R C, Chen L, Dunn P J, et al. 2000. A whole-genome assembly of Drosophila. Science, 287, 2196–2204.
Ni X Y, Tomita T, Kasai S, Kono Y. 2003. cDNA and deduced protein sequence of acetylcholinesterase from the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Applied Entomology and Zoology, 38, 49–56.
Olson D L, Christensen G M. 1980. Effects of water pollutants and other chemicals on ?sh acetylcholinesterase (in vitro). Environmental Research, 21, 327–335.
Pang Y P. 2006. Novel acetylcholinesterase target site for malaria mosquito control. PLoS One, 1, e58.
Pang Y P, Brimijoin S, Ragsdale D W, Zhu K Y, Suranyi R. 2010. Novel and viable acetylcholinesterase target site for developing effective and environmentally safe insecticides. Current Drug Targets, 13, 471–482.
Richter S, Nieveler J, Schulze H, Bachmann T T, Schmid R D. 2006. High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its puri?cation. Biotechnology and Bioengineering, 93, 1017–1022.
Scorer C A, Clare J J, McCombie W R, Romanos M A, Sreekrishna K. 1994. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. BioTechniques, 12, 181–184.
Shang J Y, Shao Y M, Lang G J, Yuan G, Tang Z H, Zhang C X. 2007. Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori, in insect cells. Insect Science, 14, 443–449.
Soderlund D M, Bloomquist J R. 1990. Molecular mechanism of insecticide resistance. In: Roush R T, Tabashnik B E, eds., Pesticide Resistance in Arthropods. Chapman and Hall, New York. pp. 58–96.
Sussman J L, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I. 1991. Atomic structure of acetylcholinesterase from Torpedo californica: A prototypic acetylcholine-binding protein. Science, 253, 872–879.
Sussman J L, Harel M, Silman I. 1993. Three-dimensional structure of acetylcholinesterase and of its complexes with anticholinesterase drugs. Chemico-Biological Interactions, 87, 187–197.
Talekar N S, Shelton A M. 1993. Biology, ecology and management of the diamondback moth. Annual Review of Entomology, 38, 275–302.
Villatte F, Bachman T T, Hussein A S, Schmid R D. 2001. Acetylcholinesterase assay for rapid expression screening in liquid and solid media. BioTechniques, 30, 81–86.
Vontas J G, Hejazi M J, Hawkes N J, Cosmidis N, Loukas M, Janes R W, Hemingway J. 2002. Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit ?y Bactrocera olea. Insect Molecular Biology, 11, 329–336.
Walsh S B, Dolden T A, Moores G, Kristensen M, Lewis T, Devonshire A L, Williamson M S. 2001. Identi?cation and characterization of mutation in the house?y (Musca domestica) acetylcholinesterase involved in insecticide resistance. Biochemical Journal, 359, 175–181.
Wang K, Liu T, Yu C, Jiang X, Yi M. 2002. Resistance of Aphis gossypii (Homoptera: Aphididae) to fenvalerate and imidacloprid and activities of detoxification enzymes on cotton and cucumber. Journal of Economic Entomology, 95, 407–413.
Wu S, Letchworth G J. 2004. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BioTechniques, 36, 152–154.
Xu S, Wu A, Chen H, Xie Y, Xu Y, Zhang L, Li J, Zhang D. 2007. Production of a novel recombinant Drosophila melanogaster acetylcholinesterase for detection of organophosphate and carbamate insecticide residues. Biomolecular Engineering, 24, 253–261.
Zhou L, Huang J, Xu H. 2011. Monitoring resistance of ?eld populations of diamondback moth Plutella xylostella L. (Lepidoptera: Yponomeutidae) to ?ve insecticides in South China: A ten-year case study. Crop Protection, 30, 272–278.
Zhou X, Xia Y. 2011. Expression and characterization of recombinant Locusta migratoria manilensis acetylcholinesterase 1 in Pichia pastoris. Protein Expression and Purification, 77, 62–67.
Zhu K Y, Lee S H, Clark J M. 1996. A point mutation of acetylcholinesterase associated with azinphosmethyl resistance and reduced fitness in Colorado potato beetle. Pesticide Biochemistry and Physiology, 55, 100–108.

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics