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A novel cry52Ca1 gene from an Indian Bacillus thuringiensis isolate is toxic to Helicoverpa armigera (cotton boll worm)
Journal of Invertebrate Pathology 159 (2018) 137–140
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
A novel cry52Ca1 gene from an Indian Bacillus thuringiensis isolate is toxic to Helicoverpa armigera (cotton boll worm) Bhupendra S. Panwara, Jaswinder Kaurb, Pradyumn Kumarb, Sarvjeet Kaura, a b
T
⁎
ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi 110012, India ICAR-Indian Institute of Maize Research, Pusa Campus, New Delhi 110012, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacillus thuringiensis Cry52Ca1 Protein expression Helicoverpa armigera
The novel cry52Ca1 gene from an Indian Bacillus thuringiensis (Bt) isolate was cloned in an expression vector (pET301/CT-DEST, 6xHis). The gene expressed as a ∼77.2 kDa protein in E. coli BL21-CodonPlus (DE3)-RIPL cells upon induction with isopropyl-thio-galactoside (IPTG) for 18 h at 28 °C. Cry52Ca1 protein was toxic to Helicoverpa armigera (cotton bollworm) neonate larvae (LC50 36.66 µg per ml and MIC50 3.051 µg per ml) in dietbased laboratory assays. This gene has potential for deployment in insect-resistant transgenic crops.
1. Introduction
2. Materials and methods
Bacillus thuringiensis (Bt) is a ubiquitous Gram-positive, sporeforming bacterium producing Crystal (Cry), Cytolytic (Cyt) and vegetative insecticidal proteins (Vip), which have specific toxicity to different insect orders. Bt has been widely used in agriculture and human health for insect pest control (Schnepf et al., 1998; Jouzani et al., 2017; Zhao et al., 2017). Over the past few decades, more than 600 different cry genes have been identified and classified into cry1 to cry77 gene families (Crickmore et al., 1998; Adang et al. 2014; Palma et al 2014). Although the cry1 gene family has been extensively used for lepidopteran insect pest control in transgenic crops, instances of development of resistance in target pests have necessitated identification of novel cry genes (Fabrick et al., 2015; Jakka et al., 2016; Rausch et al., 2016; Liu et al., 2017; Ribeiro et al., 2017; Tabashnik and Carriere, 2017). We have performed high throughput sequence analysis of native Bt isolates recovered from diverse habitats of India and identified a novel gene (National Centre for Biotechnology Information (NCBI) accession number KM053253) named as cry52Ca1 by Bacillus thuringiensis nomenclature committee (http://www.biols.susx.ac.uk/Home/Neil_ Crickmore/BT/) from a native Bt isolate SK711 (Panwar et al., 2018). In the current investigation, we have cloned this gene in an expression vector, expressed in E. coli and evaluated toxicity of Cry52Ca1 against the lepidopteran pest H. armigera.
2.1. Bacterial strains, culture condition and plasmids
⁎
E. coli strain BL21-CodonPlus (DE3)-RIPL (Invitrogen, USA) was used as a host for heterologous expression. E. coli host cells were grown at 37 °C and 200 rpm for 16 h. Plasmid DNA was isolated using alkaline lysis method (Birnboim and Doly, 1979). Quality of plasmid DNA was checked on 0.8% agarose electrophoresis (Green et al., 2012). Quantity of plasmid was estimated using UV spectrophotometer (NanoDrop, Thermo Scientific, USA). 2.2. Cloning of cry52Ca1 gene in expression vector The cry52Ca1 gene cloned into Gateway entry vector pENTR/SD/DTOPO. (Panwar et al., 2018) was mobilized into Gateway destination vector pET301/CT-DEST vector using LR clonase reaction following manufacturer's instructions (GatewayTM Cloning, Invitrogen, USA). Gene orientation and sequence were confirmed by restriction digestion and sequencing respectively. 2.3. Protein expression and purification E. coli clone carrying the recombinant pET301/CT-DEST/cry52Ca1 plasmid was grown at 37 °C in LB medium containing 100 µg per ml ampicillin until OD600 of the culture reached 0.5–0.6. The protein expression studies were performed at two temperatures for bacterial culture viz 37 °C and 28 °C. Protein expression was induced by 0.4 mM
Corresponding author. E-mail address: [email protected] (S. Kaur).
https://doi.org/10.1016/j.jip.2018.11.002 Received 28 July 2018; Received in revised form 27 October 2018; Accepted 10 November 2018 Available online 12 November 2018 0022-2011/ © 2018 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 159 (2018) 137–140
B.S. Panwar et al.
3. Results and discussion
IPTG (isopropyl-β-D-thiogalactopyranoside) and culture was incubated for 18 h. Samples were taken at 4 h and 18 h followed by lysis in 2X SDS sample buffer (100 mM Tris-HCl pH 6.8, 4% Sodium dodecyl sulphate, 0.2% bromophenol blue, 20% glycerol, 200 mM dithiothreitol) and subsequently analyzed on 12% SDS-PAGE (Sodium dodecyl sulphate–polyacrylamide gel electrophoresis). For pilot scale protein purification, the recombinant E. coli cells over-expressing Cry52Ca1-6xHis, were grown in 100 mL LB medium induced with 0.4 mM IPTG for 18 h. Thereafter, the cells were harvested by centrifugation at 15,000g, 4 °C for 10 min. The recombinant protein was purified using Ni-NTA affinity chromatography by using QIAexpress® Ni-NTA Fast Start kit and seven samples were drawn at appropriate stages of protein purification viz soluble fraction (S), pellet fraction (P), flow through fraction (FT), wash one (W1), wash two (W2), eluate 1 (E1) and eluate 2 (E2) following manufacturer's instructions. The purity of eluted fractions and quantity of recombinant protein was estimated by using spectrophotometer (NanoDrop-800, Thermo Scientific, USA) and densitometric analysis of SDS-PAGE-resolved eluted fraction.
3.1. Cloning of cry52Ca1 gene Affinity tags as a fusion partner are widely employed as a tool in protein biochemistry for the purpose of detection and purification of recombinant protein. Therefore, in order to express and purify recombinant protein, Cry52Ca1-6xHis fusion protein expression vector was constructed using Gateway recombination cloning (Rozwadowski et al., 2008). The cry52Ca1 gene is 2031 bp, encoding a polypeptide of 676 amino acids and estimated molecular mass of 77.2 kDa. To construct a 6xHis fusion protein expression vector, the gene was mobilized from the entry vector (Panwar et al., 2018) into destination vector pET301/CT-DEST using LR recombination system. The efficiency and accuracy of cloning was ensured by using both positive selection (utilizing Ampr gene) and negative selection (using ccdB gene in the destination vector). The restriction digestion should generate a linear plasmid of 7833 bp when digested with BglII or BamHI whereas, double digestion should generate two fragments of 5538 bp and 2295 bp. Restriction analysis of the recombinant vector revealed that the target gene was in the correct orientation.
2.4. Western blot analysis Protein samples resolved on 12% SDS-PAGE were transferred to the PVDF (Polyvinylidene difluoride) membrane, using electro blot system (Major Science, USA). Transfer was carried out at a constant voltage of 100 V, at 14 °C for 3 h in Towbin- SDS buffer (25 mM Tris, 192 mM glycine, 20% methanol and 0.05% SDS). Western blotting was performed using WesternBreeze® Chemiluminescent Kit (Invitrogen, USA), by following manufacturer's instructions.
3.2. Expression and solubility analysis of Cry52Ca1 protein In the current investigation, for the time course analysis, only three time points were selected, i.e. 0, 4 and 18 h after induction with IPTG, followed by incubation at 37 °C. During incubation, samples were collected at defined time intervals followed by isolation of total protein. The relative amount of protein synthesized was compared by SDS-PAGE and Western blotting. Western analysis and visual examination revealed that at 4 h after induction little or no recombinant protein was produced, whereas prolonged incubation resulted in the production of truncated protein of mass ∼25 kDa (Fig. 1a). This result indicated that prolonged incubation at 37 °C may result in the degradation of protein. Therefore, in order to minimize proteolysis of recombinant protein, the complete experiment was again performed under the lower incubation temperature of 28 °C (Fig. 1b). This decreased incubation temperature resulted in the production of full length Cry52Ca1-6xHis protein of expected molecular mass of ∼77.2 kDa after 18 h of induction. Therefore, 18 h after-induction at 28 °C was selected as a best time point for harvesting of cells for recombinant protein isolation. In addition to successful over-expression of heterologous protein in E. coli, protein solubility is another issue in production of recombinant proteins. Hence, the pilot scale experiment was performed to gather information on Cry52Ca1-6xHis protein solubility. IPTG-induced 18 h cells were harvested and protein was prepared as described in Section 2.4. All the samples collected during Ni-NTA affinity purification were analyzed by SDS-PAGE (Fig. 2a) and Western analysis (Fig. 2b). SDSPAGE and Western blot indicated some of the recombinant protein to be in the insoluble fraction while the rest was present in the soluble fraction. Recombinant protein present in the soluble fraction was easily purified by affinity chromatography. Therefore, this expression vector construct was suitable for large scale recombinant protein purification.
2.5. Protein quantification and purity analysis To quantify recombinant protein, BSA (Bovine Serum Albumin)based standard curve analysis was performed. Six BSA concentration standards viz. 0.5 µg, 1 µg, 2 µg, 3 µg, 4 µg and 5 µg were prepared. Standards and purified proteins were resolved on 12% SDS PAGE followed by Coomassie staining and de-staining by following manufacturer’s instruction (Coomassie® Blue Staining, Thermo Fisher Scientific, USA). Image was captured using gel documentation system (G:BOX, Syngene, UK) and processed with ImageJ tool available at http://rsb.info.nih.gov/ij/download.html. To quantify protein, the intensity of specific bands corresponding to BSA standards was analyzed. The intensities of bands were converted into peaks to estimate the area of the curves, which were used to plot the standard curve to generate the regression equation for quantification of recombinant protein. 2.6. Bioassay H. armigera eggs were collected from a cotton field at ICAR-Indian Agricultural Research Institute, New Delhi and maintained at ICARIndian Institute of Maize Research, New Delhi. The neonate larvae were reared in the laboratory on a semi-synthetic diet. The toxicity of Cry52Ca1 protein against neonate H. armigera larvae was evaluated by mortality bioassay using a range of 5 protein concentrations viz, 0.001 ppm, 0.01 ppm, 0.1 ppm, 1 ppm and 10 ppm in 128 well trays (CD International, Pittman, USA) with 1 mL diet dispensed per well. A single neonate larva was placed in each well. Wells were covered with perforated lids (CD International, Pittman, USA) and transferred to the control environmental chamber (25 ± 2 °C and 60–90% relative humidity, 16 h photoperiod). A total of 16 larvae were assayed per toxin concentration. The larvae on a diet without the toxin served as a control. The bioassay was replicated 3 times. After 7 days, mortality was recorded. The data were subjected to probit regression analysis and mortality was evaluated as LC50 (median lethal concentration) and MIC50 (moulting inhibition concentration). The probit regression analyses were performed using SAS package.
3.3. Protein purification and bioassay Ni-NTA affinity chromatography was utilized for protein purification. Ni-NTA chromatography separates protein via reversible interaction between the target protein and immobilized Ni+ ion attached to a chromatographic matrix. The quantity of total proteins recovered in elution was 1.25 mg as estimated by spectrophotometer. The quantification of purified protein was performed using standard curve analysis (Vincent et al., 1997). Densitometry analysis of SDS-PAGE for band intensity was performed to quantify the concentration of protein with standard curve analysis using BSA. Five BSA standards were prepared and run in 12% SDS PAGE with purified target protein. The 138
Journal of Invertebrate Pathology 159 (2018) 137–140
B.S. Panwar et al.
Fig. 1. (a) Time course analysis of protein expression at 37 °C by (i) SDS-PAGE and (ii) Western blotting. (b) Time course analysis of protein expression of Cry52Ca1 at 28 °C by (i) SDS-PAGE and (ii) Western blotting.
56% between Cry52Ba1 and Cry52Ca1 indicated that Cry52Ca1 may have a different toxicity spectrum than that of Cry52Ba1. The toxicity of Cry52Ca1 protein to H. armigera was evaluated by dose-mortality bioassay and moulting inhibition concentration (MIC) assay. The dosemortality responses of Cry52Ca1 protein for H. armigera fitted a regression line. The interface between H. armigera population and log protein concentration was significant (χ2 = 29.286, df = 16, p < 0.05), exhibiting LC50 of 36.6 ppm (95% CI, 33.87–39.45). Likewise, moulting inhibition response of Cry52Ca1 protein for H. armigera also fitted a regression line. The interface between H. armigera population and log protein concentration was significant (χ2 = 21.965,
concentration of the target protein was estimated to be 1.12 mg by densitometry. Concentration of target protein in total eluted protein indicated target protein of 89.6% purity. The purity and yield of proteins were enough to performed insect bioassay for evaluation the toxicity of Cry52Ca1 protein. Cry52 gene family is composed of three members viz Cry52Aa1, Cry52Ba1 and Cry52Ca1. Out of this only Cry52Ba1, identified from BtBM59-2, has been functionally characterized against Diptera (A. aegypti) and Lepidoptera (P. xylostella and H. armigera) with maximum toxicity against Diptera, while no toxicity towards H. armigera and P. xylostella was reported (Zheng et al., 2010). Low sequence similarity of
Fig. 2. Protein solubility analysis after 18 h of induction and incubation at 28 °C by (a) SDS-PAGE and (b) Western blotting (UT-Untransformed BL21-RIPL, CL-Crude Lysate of transformed BL21-RIPL, S-Supernatant, P-Pellet, FT-flow through, W1-First Wash, W2- Second Wash, E1- First Eluate and E2-Second Eluate). 139
Journal of Invertebrate Pathology 159 (2018) 137–140
B.S. Panwar et al.
doi.org/10.1016/j.jip.2018.11.002.
df = 16, p < 0.05), exhibiting MIC50 of 3.05 µg/ml (95% CI, 1.37–8.49). The cry52 gene family comprises of holotypes cry52Aa and cry52Ba, with one allele each: cry52Aa1 and cry52Ba1 (http://www.biols.susx. ac.uk/Home/Neil_Crickmore/BT/ accessed on 28th September 2018). Out of these, cry52Ba1 has been cloned and characterized with toxicity against Dipteran pest Aedes aegypti (Zheng et al., 2010). The novel cry52Ca1 gene, identified in our laboratory (Panwar et al., 2018), has been found to be toxic to H. armigera in this study.
References Adang, M.J., Crickmore, N., Jurat-Fuentes, J.L., 2014. Diversity of Bacillus thuringiensis crystal toxins and mechanism of action. In: Dhadialla, T.S., Gill, S.S. (Eds.), Advances in Insect Physiology 47. Academic Press, Oxford, pp. 39–87. Avilla, C., et al., 2005. Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain. J. Invertebr. Pathol. 90, 51–54. Birnboim, H.C., Doly, J., 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513–1523. Tabashnik, B.E., Carriere, Y., 2017. Surge in insect resistance to transgenic crops and prospects for sustainability. Nature Biotech. 35, 926–935. Carrière, Y., Crickmore, N., Tabashnik, B.E., 2015. Optimizing pyramided transgenic Bt crops for sustainable pest management. Nature Biotechnol. 33, 161–168. Crickmore, N., et al., 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 807–813. Fabrick, J.A., et al., 2015. Multi-toxin resistance enables pink bollworm survival on pyramided Bt cotton. Sci. Rep. 5, 16554. Green, M.R., et al., 2012. Molecular cloning : a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Jakka, S.R., et al., 2016. Broad-spectrum resistance to Bacillus thuringiensis toxins by western corn rootworm (Diabrotica virgifera virgifera). Sci. Rep. 6, 27860. Jouzani, G.S., et al., 2017. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Appl. Microbiol. Biotechnol. 101, 2691–2711. Li, H., Bouwer, G., 2012. Toxicity of Bacillus thuringiensis Cry proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) in South Africa. J. Invertebr. Pathol. 109, 110–116. Liao, C., Heckel, D.G., Akhurst, R., 2002. Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton. J. Invertebr. Pathol. 80, 55–63. Liu, L., et al., 2017. Resistance to Bacillus thuringiensis toxin Cry2Ab and survival on single-toxin and pyramided cotton in cotton bollworm from China. Evol. Appl. 10, 170–179. Palma, L., et al., 2014. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins (Basel). 6, 3296–3325. Panwar, B.S., et al., 2018. Pool deconvolution approach for high-throughput gene mining from Bacillus thuringiensis. Appl. Microbiol. Biotechnol. 102, 1467–1482. Rausch, M.A., et al., 2016. Modification of Cry4Aa toward Improved Toxin Processing in the Gut of the Pea Aphid Acyrthosiphon pisum. PLoS One 11, e0155466. Ribeiro, T.P., et al., 2017. Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil. Plant Biotechnol J. 15, 997–1009. Rozwadowski, K., et al., 2008. Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems. BMC Biotechnol. 8, 88. Schnepf, E., et al., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Vincent, S.G., et al., 1997. Quantitative densitometry of proteins stained with coomassie blue using a Hewlett Packard scanjet scanner and Scanplot software. Electrophoresis 18, 67–71. Zhao, M., et al., 2017. Functional roles of cadherin, aminopeptidase-N and alkaline phosphatase from Helicoverpa armigera (Hübner) in the action mechanism of Bacillus thuringiensis Cry2Aa. Sci. Rep. 7, 46555. Zheng, A., et al., 2010. Characterization and expression of a novel holotype insecticidal crystal protein gene from native Bacillus thuringiensis BM59-2. Can. J. Microbiol. 56, 156–161.
4. Conclusions Regardless of numerous Cry toxins having been identified and characterized, discovery of novel Cry toxins is important for further improvement of biological insecticides. In the current investigation, a novel cry52 gene family member, cry52Ca1 was cloned in an expression vector. The function characterization of Cry52Ca1 against lepidopteran pest H. armigera revealed LC50 of 36.66 ppm and MIC50 of 3.051 µg per ml. Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab and Vip3A proteins are the other Bt toxins reported to be active against H. armigera, out of which, Cry1Ac (LC50 ranging from 115 to 248 ng per cm2) and Cry2Aa (LC50 149 ng per cm2) are reported to be most toxic against Australian population of H. armigera (Liao et al., 2002). Cry1Ac (LC50 3.4 µg per ml) and Cry2Aa (LC50 6.3 µg per ml) are also more toxic, as compared with other Bt toxins, towards Spanish (Avilla et al., 2005) and South African populations of H. armigera (Li and Bouwer, 2012). In view of development of resistance in H. armigera to the widely used cry1Ac and cry2A genes in transgenic crops (Carrière et al., 2015), the cry52Ca1 gene has potential for deployment in insect pest control due to its low structural homology with these genes (Panwar et al., 2018). Acknowledgement BSP acknowledges the University Grants Commission (UGC), Government of India for providing junior research fellowship. We also acknowledge ICAR-National Research Centre on Plant Biotechnology for providing the infrastructure and facilities for carrying out these studies. This work is a part of Ph.D. thesis of BSP submitted to PostGraduate School, ICAR-IARI, New Delhi, India. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Appendix A. Supplementary material Supplementary data to this article can be found online at https://
140
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
A novel cry52Ca1 gene from an Indian Bacillus thuringiensis isolate is toxic to Helicoverpa armigera (cotton boll worm) Bhupendra S. Panwara, Jaswinder Kaurb, Pradyumn Kumarb, Sarvjeet Kaura, a b
T
⁎
ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi 110012, India ICAR-Indian Institute of Maize Research, Pusa Campus, New Delhi 110012, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacillus thuringiensis Cry52Ca1 Protein expression Helicoverpa armigera
The novel cry52Ca1 gene from an Indian Bacillus thuringiensis (Bt) isolate was cloned in an expression vector (pET301/CT-DEST, 6xHis). The gene expressed as a ∼77.2 kDa protein in E. coli BL21-CodonPlus (DE3)-RIPL cells upon induction with isopropyl-thio-galactoside (IPTG) for 18 h at 28 °C. Cry52Ca1 protein was toxic to Helicoverpa armigera (cotton bollworm) neonate larvae (LC50 36.66 µg per ml and MIC50 3.051 µg per ml) in dietbased laboratory assays. This gene has potential for deployment in insect-resistant transgenic crops.
1. Introduction
2. Materials and methods
Bacillus thuringiensis (Bt) is a ubiquitous Gram-positive, sporeforming bacterium producing Crystal (Cry), Cytolytic (Cyt) and vegetative insecticidal proteins (Vip), which have specific toxicity to different insect orders. Bt has been widely used in agriculture and human health for insect pest control (Schnepf et al., 1998; Jouzani et al., 2017; Zhao et al., 2017). Over the past few decades, more than 600 different cry genes have been identified and classified into cry1 to cry77 gene families (Crickmore et al., 1998; Adang et al. 2014; Palma et al 2014). Although the cry1 gene family has been extensively used for lepidopteran insect pest control in transgenic crops, instances of development of resistance in target pests have necessitated identification of novel cry genes (Fabrick et al., 2015; Jakka et al., 2016; Rausch et al., 2016; Liu et al., 2017; Ribeiro et al., 2017; Tabashnik and Carriere, 2017). We have performed high throughput sequence analysis of native Bt isolates recovered from diverse habitats of India and identified a novel gene (National Centre for Biotechnology Information (NCBI) accession number KM053253) named as cry52Ca1 by Bacillus thuringiensis nomenclature committee (http://www.biols.susx.ac.uk/Home/Neil_ Crickmore/BT/) from a native Bt isolate SK711 (Panwar et al., 2018). In the current investigation, we have cloned this gene in an expression vector, expressed in E. coli and evaluated toxicity of Cry52Ca1 against the lepidopteran pest H. armigera.
2.1. Bacterial strains, culture condition and plasmids
⁎
E. coli strain BL21-CodonPlus (DE3)-RIPL (Invitrogen, USA) was used as a host for heterologous expression. E. coli host cells were grown at 37 °C and 200 rpm for 16 h. Plasmid DNA was isolated using alkaline lysis method (Birnboim and Doly, 1979). Quality of plasmid DNA was checked on 0.8% agarose electrophoresis (Green et al., 2012). Quantity of plasmid was estimated using UV spectrophotometer (NanoDrop, Thermo Scientific, USA). 2.2. Cloning of cry52Ca1 gene in expression vector The cry52Ca1 gene cloned into Gateway entry vector pENTR/SD/DTOPO. (Panwar et al., 2018) was mobilized into Gateway destination vector pET301/CT-DEST vector using LR clonase reaction following manufacturer's instructions (GatewayTM Cloning, Invitrogen, USA). Gene orientation and sequence were confirmed by restriction digestion and sequencing respectively. 2.3. Protein expression and purification E. coli clone carrying the recombinant pET301/CT-DEST/cry52Ca1 plasmid was grown at 37 °C in LB medium containing 100 µg per ml ampicillin until OD600 of the culture reached 0.5–0.6. The protein expression studies were performed at two temperatures for bacterial culture viz 37 °C and 28 °C. Protein expression was induced by 0.4 mM
Corresponding author. E-mail address: [email protected] (S. Kaur).
https://doi.org/10.1016/j.jip.2018.11.002 Received 28 July 2018; Received in revised form 27 October 2018; Accepted 10 November 2018 Available online 12 November 2018 0022-2011/ © 2018 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 159 (2018) 137–140
B.S. Panwar et al.
3. Results and discussion
IPTG (isopropyl-β-D-thiogalactopyranoside) and culture was incubated for 18 h. Samples were taken at 4 h and 18 h followed by lysis in 2X SDS sample buffer (100 mM Tris-HCl pH 6.8, 4% Sodium dodecyl sulphate, 0.2% bromophenol blue, 20% glycerol, 200 mM dithiothreitol) and subsequently analyzed on 12% SDS-PAGE (Sodium dodecyl sulphate–polyacrylamide gel electrophoresis). For pilot scale protein purification, the recombinant E. coli cells over-expressing Cry52Ca1-6xHis, were grown in 100 mL LB medium induced with 0.4 mM IPTG for 18 h. Thereafter, the cells were harvested by centrifugation at 15,000g, 4 °C for 10 min. The recombinant protein was purified using Ni-NTA affinity chromatography by using QIAexpress® Ni-NTA Fast Start kit and seven samples were drawn at appropriate stages of protein purification viz soluble fraction (S), pellet fraction (P), flow through fraction (FT), wash one (W1), wash two (W2), eluate 1 (E1) and eluate 2 (E2) following manufacturer's instructions. The purity of eluted fractions and quantity of recombinant protein was estimated by using spectrophotometer (NanoDrop-800, Thermo Scientific, USA) and densitometric analysis of SDS-PAGE-resolved eluted fraction.
3.1. Cloning of cry52Ca1 gene Affinity tags as a fusion partner are widely employed as a tool in protein biochemistry for the purpose of detection and purification of recombinant protein. Therefore, in order to express and purify recombinant protein, Cry52Ca1-6xHis fusion protein expression vector was constructed using Gateway recombination cloning (Rozwadowski et al., 2008). The cry52Ca1 gene is 2031 bp, encoding a polypeptide of 676 amino acids and estimated molecular mass of 77.2 kDa. To construct a 6xHis fusion protein expression vector, the gene was mobilized from the entry vector (Panwar et al., 2018) into destination vector pET301/CT-DEST using LR recombination system. The efficiency and accuracy of cloning was ensured by using both positive selection (utilizing Ampr gene) and negative selection (using ccdB gene in the destination vector). The restriction digestion should generate a linear plasmid of 7833 bp when digested with BglII or BamHI whereas, double digestion should generate two fragments of 5538 bp and 2295 bp. Restriction analysis of the recombinant vector revealed that the target gene was in the correct orientation.
2.4. Western blot analysis Protein samples resolved on 12% SDS-PAGE were transferred to the PVDF (Polyvinylidene difluoride) membrane, using electro blot system (Major Science, USA). Transfer was carried out at a constant voltage of 100 V, at 14 °C for 3 h in Towbin- SDS buffer (25 mM Tris, 192 mM glycine, 20% methanol and 0.05% SDS). Western blotting was performed using WesternBreeze® Chemiluminescent Kit (Invitrogen, USA), by following manufacturer's instructions.
3.2. Expression and solubility analysis of Cry52Ca1 protein In the current investigation, for the time course analysis, only three time points were selected, i.e. 0, 4 and 18 h after induction with IPTG, followed by incubation at 37 °C. During incubation, samples were collected at defined time intervals followed by isolation of total protein. The relative amount of protein synthesized was compared by SDS-PAGE and Western blotting. Western analysis and visual examination revealed that at 4 h after induction little or no recombinant protein was produced, whereas prolonged incubation resulted in the production of truncated protein of mass ∼25 kDa (Fig. 1a). This result indicated that prolonged incubation at 37 °C may result in the degradation of protein. Therefore, in order to minimize proteolysis of recombinant protein, the complete experiment was again performed under the lower incubation temperature of 28 °C (Fig. 1b). This decreased incubation temperature resulted in the production of full length Cry52Ca1-6xHis protein of expected molecular mass of ∼77.2 kDa after 18 h of induction. Therefore, 18 h after-induction at 28 °C was selected as a best time point for harvesting of cells for recombinant protein isolation. In addition to successful over-expression of heterologous protein in E. coli, protein solubility is another issue in production of recombinant proteins. Hence, the pilot scale experiment was performed to gather information on Cry52Ca1-6xHis protein solubility. IPTG-induced 18 h cells were harvested and protein was prepared as described in Section 2.4. All the samples collected during Ni-NTA affinity purification were analyzed by SDS-PAGE (Fig. 2a) and Western analysis (Fig. 2b). SDSPAGE and Western blot indicated some of the recombinant protein to be in the insoluble fraction while the rest was present in the soluble fraction. Recombinant protein present in the soluble fraction was easily purified by affinity chromatography. Therefore, this expression vector construct was suitable for large scale recombinant protein purification.
2.5. Protein quantification and purity analysis To quantify recombinant protein, BSA (Bovine Serum Albumin)based standard curve analysis was performed. Six BSA concentration standards viz. 0.5 µg, 1 µg, 2 µg, 3 µg, 4 µg and 5 µg were prepared. Standards and purified proteins were resolved on 12% SDS PAGE followed by Coomassie staining and de-staining by following manufacturer’s instruction (Coomassie® Blue Staining, Thermo Fisher Scientific, USA). Image was captured using gel documentation system (G:BOX, Syngene, UK) and processed with ImageJ tool available at http://rsb.info.nih.gov/ij/download.html. To quantify protein, the intensity of specific bands corresponding to BSA standards was analyzed. The intensities of bands were converted into peaks to estimate the area of the curves, which were used to plot the standard curve to generate the regression equation for quantification of recombinant protein. 2.6. Bioassay H. armigera eggs were collected from a cotton field at ICAR-Indian Agricultural Research Institute, New Delhi and maintained at ICARIndian Institute of Maize Research, New Delhi. The neonate larvae were reared in the laboratory on a semi-synthetic diet. The toxicity of Cry52Ca1 protein against neonate H. armigera larvae was evaluated by mortality bioassay using a range of 5 protein concentrations viz, 0.001 ppm, 0.01 ppm, 0.1 ppm, 1 ppm and 10 ppm in 128 well trays (CD International, Pittman, USA) with 1 mL diet dispensed per well. A single neonate larva was placed in each well. Wells were covered with perforated lids (CD International, Pittman, USA) and transferred to the control environmental chamber (25 ± 2 °C and 60–90% relative humidity, 16 h photoperiod). A total of 16 larvae were assayed per toxin concentration. The larvae on a diet without the toxin served as a control. The bioassay was replicated 3 times. After 7 days, mortality was recorded. The data were subjected to probit regression analysis and mortality was evaluated as LC50 (median lethal concentration) and MIC50 (moulting inhibition concentration). The probit regression analyses were performed using SAS package.
3.3. Protein purification and bioassay Ni-NTA affinity chromatography was utilized for protein purification. Ni-NTA chromatography separates protein via reversible interaction between the target protein and immobilized Ni+ ion attached to a chromatographic matrix. The quantity of total proteins recovered in elution was 1.25 mg as estimated by spectrophotometer. The quantification of purified protein was performed using standard curve analysis (Vincent et al., 1997). Densitometry analysis of SDS-PAGE for band intensity was performed to quantify the concentration of protein with standard curve analysis using BSA. Five BSA standards were prepared and run in 12% SDS PAGE with purified target protein. The 138
Journal of Invertebrate Pathology 159 (2018) 137–140
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Fig. 1. (a) Time course analysis of protein expression at 37 °C by (i) SDS-PAGE and (ii) Western blotting. (b) Time course analysis of protein expression of Cry52Ca1 at 28 °C by (i) SDS-PAGE and (ii) Western blotting.
56% between Cry52Ba1 and Cry52Ca1 indicated that Cry52Ca1 may have a different toxicity spectrum than that of Cry52Ba1. The toxicity of Cry52Ca1 protein to H. armigera was evaluated by dose-mortality bioassay and moulting inhibition concentration (MIC) assay. The dosemortality responses of Cry52Ca1 protein for H. armigera fitted a regression line. The interface between H. armigera population and log protein concentration was significant (χ2 = 29.286, df = 16, p < 0.05), exhibiting LC50 of 36.6 ppm (95% CI, 33.87–39.45). Likewise, moulting inhibition response of Cry52Ca1 protein for H. armigera also fitted a regression line. The interface between H. armigera population and log protein concentration was significant (χ2 = 21.965,
concentration of the target protein was estimated to be 1.12 mg by densitometry. Concentration of target protein in total eluted protein indicated target protein of 89.6% purity. The purity and yield of proteins were enough to performed insect bioassay for evaluation the toxicity of Cry52Ca1 protein. Cry52 gene family is composed of three members viz Cry52Aa1, Cry52Ba1 and Cry52Ca1. Out of this only Cry52Ba1, identified from BtBM59-2, has been functionally characterized against Diptera (A. aegypti) and Lepidoptera (P. xylostella and H. armigera) with maximum toxicity against Diptera, while no toxicity towards H. armigera and P. xylostella was reported (Zheng et al., 2010). Low sequence similarity of
Fig. 2. Protein solubility analysis after 18 h of induction and incubation at 28 °C by (a) SDS-PAGE and (b) Western blotting (UT-Untransformed BL21-RIPL, CL-Crude Lysate of transformed BL21-RIPL, S-Supernatant, P-Pellet, FT-flow through, W1-First Wash, W2- Second Wash, E1- First Eluate and E2-Second Eluate). 139
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doi.org/10.1016/j.jip.2018.11.002.
df = 16, p < 0.05), exhibiting MIC50 of 3.05 µg/ml (95% CI, 1.37–8.49). The cry52 gene family comprises of holotypes cry52Aa and cry52Ba, with one allele each: cry52Aa1 and cry52Ba1 (http://www.biols.susx. ac.uk/Home/Neil_Crickmore/BT/ accessed on 28th September 2018). Out of these, cry52Ba1 has been cloned and characterized with toxicity against Dipteran pest Aedes aegypti (Zheng et al., 2010). The novel cry52Ca1 gene, identified in our laboratory (Panwar et al., 2018), has been found to be toxic to H. armigera in this study.
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4. Conclusions Regardless of numerous Cry toxins having been identified and characterized, discovery of novel Cry toxins is important for further improvement of biological insecticides. In the current investigation, a novel cry52 gene family member, cry52Ca1 was cloned in an expression vector. The function characterization of Cry52Ca1 against lepidopteran pest H. armigera revealed LC50 of 36.66 ppm and MIC50 of 3.051 µg per ml. Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab and Vip3A proteins are the other Bt toxins reported to be active against H. armigera, out of which, Cry1Ac (LC50 ranging from 115 to 248 ng per cm2) and Cry2Aa (LC50 149 ng per cm2) are reported to be most toxic against Australian population of H. armigera (Liao et al., 2002). Cry1Ac (LC50 3.4 µg per ml) and Cry2Aa (LC50 6.3 µg per ml) are also more toxic, as compared with other Bt toxins, towards Spanish (Avilla et al., 2005) and South African populations of H. armigera (Li and Bouwer, 2012). In view of development of resistance in H. armigera to the widely used cry1Ac and cry2A genes in transgenic crops (Carrière et al., 2015), the cry52Ca1 gene has potential for deployment in insect pest control due to its low structural homology with these genes (Panwar et al., 2018). Acknowledgement BSP acknowledges the University Grants Commission (UGC), Government of India for providing junior research fellowship. We also acknowledge ICAR-National Research Centre on Plant Biotechnology for providing the infrastructure and facilities for carrying out these studies. This work is a part of Ph.D. thesis of BSP submitted to PostGraduate School, ICAR-IARI, New Delhi, India. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Appendix A. Supplementary material Supplementary data to this article can be found online at https://
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