High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application
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 concentrati...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2017-06-01
|
| Series: | Journal of Integrative Agriculture |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2095311916615083 |
| _version_ | 1818893149727096832 |
|---|---|
| author | Niu LIU Jun CHE Duo LAI Jin-jun WEN Han-hong XU |
| author_facet | Niu LIU Jun CHE Duo LAI Jin-jun WEN Han-hong XU |
| author_sort | Niu LIU |
| collection | DOAJ |
| description | 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 first precipitated with ammonium sulfate (50% saturation) and then purified with procainamide affinity column chromatography. The enzyme was purified 12.1-fold with a yield of 22.8% and a high specific 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. |
| first_indexed | 2024-12-19T18:08:01Z |
| format | Article |
| id | doaj.art-ffa9d32d9de74d0c88bebd92043d9639 |
| institution | Directory Open Access Journal |
| issn | 2095-3119 |
| language | English |
| last_indexed | 2024-12-19T18:08:01Z |
| publishDate | 2017-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Integrative Agriculture |
| spelling | doaj.art-ffa9d32d9de74d0c88bebd92043d96392022-12-21T20:11:24ZengElsevierJournal of Integrative Agriculture2095-31192017-06-0116613581366High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential applicationNiu LIU0Jun CHE1Duo LAI2Jin-jun WEN3Han-hong XU4State 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.ChinaState 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; Shenzhen Longgang Inspection Station of Non-pollution Agricultural Products, Shenzhen 510172, P.R.ChinaState 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; Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.ChinaState 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.ChinaState 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; Correspondence XU Han-hong, Tel: +86-20-85285127The 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 first precipitated with ammonium sulfate (50% saturation) and then purified with procainamide affinity column chromatography. The enzyme was purified 12.1-fold with a yield of 22.8% and a high specific 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.http://www.sciencedirect.com/science/article/pii/S2095311916615083Plutella xylostellaacetylcholinesterasePichia pastorishigh-level expression |
| spellingShingle | Niu LIU Jun CHE Duo LAI Jin-jun WEN Han-hong XU High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application Journal of Integrative Agriculture Plutella xylostella acetylcholinesterase Pichia pastoris high-level expression |
| title | High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application |
| title_full | High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application |
| title_fullStr | High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application |
| title_full_unstemmed | High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application |
| title_short | High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application |
| title_sort | high level expression and purification of plutella xylostella acetylcholinesterase in pichia pastoris and its potential application |
| topic | Plutella xylostella acetylcholinesterase Pichia pastoris high-level expression |
| url | http://www.sciencedirect.com/science/article/pii/S2095311916615083 |
| work_keys_str_mv | AT niuliu highlevelexpressionandpurificationofplutellaxylostellaacetylcholinesteraseinpichiapastorisanditspotentialapplication AT junche highlevelexpressionandpurificationofplutellaxylostellaacetylcholinesteraseinpichiapastorisanditspotentialapplication AT duolai highlevelexpressionandpurificationofplutellaxylostellaacetylcholinesteraseinpichiapastorisanditspotentialapplication AT jinjunwen highlevelexpressionandpurificationofplutellaxylostellaacetylcholinesteraseinpichiapastorisanditspotentialapplication AT hanhongxu highlevelexpressionandpurificationofplutellaxylostellaacetylcholinesteraseinpichiapastorisanditspotentialapplication |