Expression and Purification cry1Aa Gene

Enny Rimita Sembiring, Agus Rachmat, Wien Kusharyoto, Puspo Edi Giriwono, Satya Nugroho, Maggy Thenawidjaja Suhartono

Abstract


The cry gene encodes a crystal protein of Bacillus thuringiensis known as Cry toxin, which is toxic to major Lepidopteran insect pest. The effectiveness of Cry toxin against the rice yellow stem borer (YSB, Scirpophaga incertulas Wlk) has been reported. Transgenic Bacillus thuringiensis (Bt) rice has been developed by introducing the fusion gene cry1B::cry1Aa gene driven by the CaMV35S promoter to Javanica rice cv. Rojolele to improve its resistance against rice YSB. Resistance to YSB at a greenhouse and multi-site scales have been conducted and reported. Food and feed safety studies for the release and commercialization of genetically modified crops require a sufficient amount of purified protein sample. Therefore, a strategy to produce Cry1Aa protein in a short time with high productivity needs to be developed. In this study, the cry1Aa gene (1857 bp) was cloned into pRHA-SDM expression vector under the control of the pRHA promoter fused to a 6xHis tag on the C-terminal to produce pRHA-SDM-cry1Aa. The expression host used was Escherichia coli strain NiCo21, and protein purification was performed using IMAC Co2+ on the TALON® matrix. The results showed that the recombinant Cry1Aa protein, approximately 69 kDa, was detected using Western blot performed using anti-rabbit Cry1Aa polyclonal antibody and His detector nickel-NTA conjugated with HRP reporter enzyme. Expression and purification protocols that have been developed can be used to produce Cry1Aa protein which can be utilized for further protein studies.

Keywords


Bacillus thuringiensis; cloning; cry1Aa; expression; purification.

Full Text:

PDF

References


A. K. Saxena, M. Kumar, H. Chakdar, N. Anuroopa, and D. J. Bagyaraj, “Bacillus species in soil as a natural resource for plant health and nutrition,” J. Appl. Microbiol., vol. 128, no. 6, pp. 1583–1594, 2020.

H. Loutfi et al., “Morphological Study of Bacillus thuringiensis Crystals and Spores,” Appl. Sci., vol. 11, no. 1, p. 155, 2020.

M. Domínguez-Arrizabalaga, M. Villanueva, B. Escriche, C. Ancín-Azpilicueta, and P. Caballero, “Insecticidal activity of bacillus thuringiensis proteins against coleopteran pests,” Toxins (Basel)., vol. 12, no. 7, p. 430, 2020.

K. Kumar et al., “Genetically modified crops: current status and future prospects,” Planta, vol. 251, no. 4. Springer, p. 91, 2020.

R. Erasmus et al., “Introgression of a cry1Ab transgene into open pollinated maize and its effect on Cry protein concentration and target pest survival,” PLoS One, vol. 14, no. 12, p. e0226476, 2019.

Y. Song et al., “Effects of transgenic Bt rice lines with single Cry1Ab and fused Cry1Ab/Cry1Ac on the abundance dynamics and community diversity of soil mites,” Arch. Agron. Soil Sci., vol. 66, no. 5, pp. 586–599, 2020.

B. Hajimohammadi et al., “Safety assessment of genetically modified rice expressing Cry1Ab protein in Sprague–Dawley rats,” Sci. Rep., vol. 11, no. 1, p. 1126, 2021.

L. Niu et al., “Transgenic Bt rice lines producing Cry1Ac, Cry2Aa or Cry1Ca have no detrimental effects on Brown Planthopper and Pond Wolf Spider,” Sci. Rep., vol. 7, no. 1, pp. 1–7, 2017.

Y. Sun et al., “Knockdown of the aminopeptidase N genes decreases susceptibility of Chilo suppressalis larvae to Cry1Ab/Cry1Ac and Cry1Ca,” Pestic. Biochem. Physiol., vol. 162, pp. 36–42, 2020.

X. Chang et al., “Cry1C rice doesn’t affect the ecological fitness of rice brown planthopper, Nilaparvata lugens either under RDV stress or not,” Sci. Rep., vol. 10, no. 1, p. 16423, 2020.

Y. Yang, B. Zhang, X. Zhou, J. Romeis, Y. Peng, and Y. Li, “Toxicological and Biochemical Analyses Demonstrate the Absence of Lethal or Sublethal Effects of cry1C- or cry2A-Expressing Bt Rice on the Collembolan Folsomia candida,” Front. Plant Sci., vol. 9, p. 131, 2018.

C. Dang, X. Zhou, C. Sun, F. Wang, Y. Peng, and G. Ye, “Impacts of Bt rice on non-target organisms assessed by the hazard quotient (HQ),” Ecotoxicol. Environ. Saf., vol. 207, p. 111214, 2021.

L. Liu, S. Knauth, L. Wu, and T. Eickhorst, “Cry1Ab/Ac proteins released from subspecies of Bacillus thuringiensis (Bt) and transgenic Bt-rice in different paddy soils,” Arch. Agron. Soil Sci., vol. 66, no. 11, pp. 1546–1555, Sep. 2020.

D. Boddupally, S. Tamirisa, S. R. Gundra, D. R. Vudem, and V. R. Khareedu, “Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests,” Sci. Rep., vol. 8, no. 1, p. 8458, 2018.

S. Nugroho, I. H. Slamet-Loedin, and A. Estiati, “Development of yellow stem borer resistant rice varieties in Indonesia,” in Genetically Modified Crops in Asia Pacific, G. Gujar, Y. Trisyono, and M. Chen, Eds. C SIRO Publishing, C SIRO Australia, 2021, pp. 245–254.

S. Rahmawati, D. Astuti, and I. H. S-loedin, “Agrobacterium-Mediated Transformation of Javanica Rice Plants with A Cry1B Gene Under The Control of Wound-Inducible Gene Promoter,” Ann. Bogor., vol. II, no. 1, pp. 1–5, 2007.

Y. Sulistyowati, A. Rachmat, F. Zahra, and S. Rahmawati, “Genetic transformation of rice cv . Ciherang using double T-DNA vector harboring cry 1Ab gene,” vol. 15, no. 1, pp. 27–32, 2011.

S. Rahmawati and I. H. Slamet-Loedin, “Introduksi Gen cryIB-cryIAa ke dalam Genom Padi (Oryza sativa) cv. Rojolele Menggunakan Transformasi Agrobacterium,” HAYATI J. Biosci., vol. 13, no. 1, pp. 19–25, 2006.

N. Usyati, D. Buchori, S. Manuwoto, P. Hidayat, and I. H. S. -Loedin, “Keefektivan padi transgenik terhadap hama penggerek batang padi kuning Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae),” J. Entomol. Indones., vol. 6, no. 1, p. 30, 2016.

A. Estiati, A. Nena, and S. Nugroho, “Insect Bioassay in Biosafety Containment to Select Transgenic Rice ( Oryza sativa L .) Harboring Cry1B Gene Resistant to Yellow Stem Borer ( Scrirpophaga incertulas Walk .),” Ann. Bogor., vol. 17, no. 2, pp. 17–26, 2013.

S. Nugroho, D. I. Sari, F. Zahra, S. Rachmawati, B. S. Maulana, and A. Estiati, “Resistant performance of T10 Rojolele transgenic rice events harboring cry1B :: cry1Aa fusion genes against the rice yellow stem borer Scirpophaga incertulas Wlk,” IOP Conf. Ser. Earth Environ. Sci., vol. 481, no. 1, 2020.

P. D. Dwivedi, M. Das, S. Kumar, and A. K. Verma, Safety assessment of food derived from genetically modified crops. Academic Press Inc., 2020.

S. Schillberg, N. Raven, H. Spiegel, S. Rasche, and M. Buntru, “Critical analysis of the commercial potential of plants for the production of recombinant proteins,” Front. Plant Sci., vol. 10, no. June, 2019.

G. L. Rosano, E. S. Morales, and E. A. Ceccarelli, “New tools for recombinant protein production in Escherichia coli : A 5‐year update,” Protein Sci., vol. 28, no. 8, pp. 1412–1422, 2019.

Roberts and C. S., “A comprehensive set of modular vectors for advanced manipulations and efficient transformation of plants.,” Rockefeller Found. Meet. Int. Progr. Rice Biotechnol., 1997.

Hariyatun, A. Suwanto, and W. Kusharyoto, “Expression of An Immunogenic Intimin Fragment of EHEC O157:H7 in Escherichia coli Periplasm under The Control of A Rhamnose-Based Regulated Promoter,” Ann. Bogor., vol. 18, no. 1, pp. 25–34, 2014.

Y. Angela, V. Wy, and A. Julius, “Preparation of Calcium Competent Escherichia coli and Heat-Shock Transformation | jemi.microbiology.ubc.ca,” Vancouver, Canada, 2017.

A. Karyolaimos et al., “Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening,” Front. Microbiol., vol. 10, p. 1511, 2019.

J. Li, X. Zhang, M. Ashokkumar, D. Liu, and T. Ding, “Molecular regulatory mechanisms of Escherichia coli O157:H7 in response to ultrasonic stress revealed by proteomic analysis,” Ultrason. Sonochem., vol. 61, p. 104835, 2020.

I. Ahmad et al., “Overcoming challenges for amplified expression of recombinant proteins using Escherichia coli,” Protein Expr. Purif., vol. 144, no. October 2017, pp. 12–18, 2018.

A. Wegerer, T. Sun, and J. Altenbuchner, “Optimization of an E. coli L-rhamnose-inducible expression vector: Test of various genetic module combinations,” BMC Biotechnol., vol. 8, no. 1, pp. 1–12, 2008.

P. D. Riggs, “Overview of Protein Expression Vectors for E. coli,” Curr. Protoc. Essent. Lab. Tech., vol. 17, no. 1, p. e23, 2018.

J. Schröder, “Development and Optimization of Purification Processes and Protein Stabilization Methods for Enzymes Applied for Biocatalytical N-Acetylneuraminic Acid Synthesis,” Haw Hamburg University, Germany, 2020.

C. Robichon, J. Luo, T. B. Causey, J. S. Benner, and J. C. Samuelson, “Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli Protein contamination after purification by immobilized metal affinity chromatography,” Appl. Environ. Microbiol., vol. 77, no. 13, pp. 4634–4646, 2011.

A. Tarar, E. M. Alyami, and C.-A. Peng, “Efficient Expression of Soluble Recombinant Protein Fused with Core-Streptavidin in Bacterial Strain with T7 Expression System,” Methods Protoc., vol. 3, no. 4, p. 82, 2020.

N. Niesler, J. Arndt, K. Silberreis, and H. Fuchs, “Generation of a soluble and stable apoptin-EGF fusion protein, a targeted viral protein applicable for tumor therapy,” Protein Expr. Purif., vol. 175, p. 105687, 2020.

T. Von Trotha, R. Jöhr, J. Fischer, L. C. Schendel, H. E. Gaub, and C. Kluger, “Kinetic Interval Measurement: A Tool to Characterize Thermal Reversion Dynamics of Light-switchable Fluorescent Proteins,” bioRxiv, 2021.

L. Soini, S. Leysen, J. Davis, M. Westwood, and C. Ottmann, “The 14‐3‐3/SLP76 protein–protein interaction in T‐cell receptor signalling: a structural and biophysical characterization,” FEBS Lett., vol. 595, no. 3, pp. 404–414, 2021.

S. Wang et al., “Expression and purification of an FGF9 fusion protein in E. coli, and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration,” Appl. Microbiol. Biotechnol., vol. 101, no. 21, pp. 7823–7835, 2017.

L. Pillai-Kastoori, A. R. Schutz-Geschwender, and J. A. Harford, “A systematic approach to quantitative Western blot analysis,” Analytical Biochemistry, vol. 593. Academic Press Inc., p. 113608, 2020.

D. H. Sauka and G. B. Benintende, “Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina,” Rev. Argent. Microbiol., vol. 49, no. 3, pp. 273–281, 2017.

M. El Khoury, H. Azzouz, A. Chavanieu, N. Abdelmalak, J. Chopineau, and M. K. Awad, “Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae,” Arch. Microbiol., vol. 196, no. 6, pp. 435–444, 2014.




DOI: http://dx.doi.org/10.18517/ijaseit.12.2.15022

Refbacks

  • There are currently no refbacks.



Published by INSIGHT - Indonesian Society for Knowledge and Human Development