Preparation and purification of Cry1Ah protein candidate reference material.

Lin Guo; Lili Geng; Xiaoxiao Sun; Meiling Wang; Changlong Shu; Jie Zhang

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Preparation and purification of Cry1Ah protein candidate reference material.

Author: Lin GUO ¹ ; Lili GENG ² ; Xiaoxiao SUN ² ; Meiling WANG ² ; Changlong SHU ² ; Jie ZHANG ²
Author Information

1. Faculty of Agronomy, Jilin Agricultural University, Changchun 130118, Jilin, China.
2. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Publication Type:Journal Article
Keywords: Bacillus thuringiensis; Cry1Ah protein; certified reference material; genetically modified organisms
MeSH: Animals; Bacillus thuringiensis; Bacterial Proteins; Cryptochromes; metabolism; Endotoxins; Hemolysin Proteins; Moths; Pest Control, Biological; Plants, Genetically Modified
From: Chinese Journal of Biotechnology 2019;35(8):1511-1519
CountryChina
Language:Chinese
Abstract: With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.