Easy and Non-Expensive Method to Increase the Production, Enhance the Stability and Improve the Purification of Cry1Ab Crystals

Rosalía Contreras¹, Christian Hernández¹, Manuel Acosta¹, Viviana Pitones², Gretel Mendoza³ & Jorge Olmos^1*

¹Molecular Microbiology Laboratory, Department of Marine Biotechnology, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, B.C., México
²Faculty of Dentistry, Universidad Autónoma de Baja California, Mexicali, BC, México
³Academic Unit of Biological Sciences, Universidad Autónoma de Zacatecas, Zacatecas, México

*Correspondence to: Dr. Jorge Olmos, Molecular Microbiology Laboratory, Department of Marine Biotechnology, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, B.C., México.

Copyright © 2018 Dr. Muhammad Sarwar Khan, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
Bacillus thuringiensis (Bt) is a Gram-positive spore forming bacterium with great importance to agriculture because the insecticidal activity produced by its Cry proteins; also known as δ-endotoxins [1,2]. Insecticidal activity is principally induced by Cry proteins specific interaction with the membrane receptors of midgut epithelium cells of insects [3]. In this sense, increasing Cry proteins production, stability and purification changing culture conditions, will facilitate the assays development on insect and human cell lines [4]. Cry proteins are produced as crystal inclusion bodies (CIB) through the Bt sporulation process. CIB production levels depends on Bt strain selected, culture medium ingredients utilized and growth conditions established [5]. Its purification has been extensively reported, however most methods are expensive and time consuming, because the physical and chemical similarities between CIB and spores [6,7]. Hence, to improve CIB production, stability and purification without affecting their insecticidal activity; medium ingredients, growth conditions and a simple filtration procedure were evaluated. Thus, several carbon:nitrogen (C7:N1, C3.5:N4.5, C1:N7) nutrient ratios were assayed to evaluate their influence on the Cry proteins production and stability. However, it is reported that a batch fermentation using Bt and high carbon concentrations (C7:N1) may decrease lower than five units the pH, affecting the bacterium development [8]. On the other hand, high nitrogen concentrations as those found in C1:N7 ratio; could increase the pH over nine units decreasing the bacterial development and the crystal stability [1,9].

Material and Method

Culture Mediums and Bt Growth
In this work 600ml of C3.5:N4.5 medium containing; 2.0gr/L of sucrose, 1.5gr/L of corn syrup, 4.0gr/L of peptone and 0.5gr/L of yeast extract, were used to growth a Bt recombinant strain (BtRS) harboring the pHT315-1Ab plasmid, which contains the Cry1Ab gene [10]. The BtRS growth and its Cry1Ab crystal production in C3.5:N4.5 medium was compared with C7:N1 medium containing 7gr/lt of sucrose and 1gr/l of peptone and with C1:N7 medium, which contains 1gr/lt of sucrose and 7gr/lt of peptone. In addition, 0.250gr/L of MgSO₄ and 1g/L of KCl were added to all mediums, pH was adjusted to 7.0 and mediums were sterilized. Subsequently, 2ml/L at 10mM of MnCl₂, 1ml/L at 0.5M of CaCl₂, 1ml/L at 0.15M of FeSO₄ and 25µg/ml of erythromycin were supplemented. Growth conditions were settled at 200rpm and 30^oC using Erlenmeyer flasks of 1000ml. BtRS strain was grown 72 h in 600ml of each medium, samples were taken each hour during exponential growth and every 12 h through the sporulation phase.

CIB Purification
The BtRS strain was grown 72 h in 200ml of C3.5:N4.5 medium, this volume was centrifuged at 8000 x g by 10 min and pellet was resuspended in 200ml of TBE buffer pH 8.5 to maintain CIB stability. Resuspended CIB were passed through a 0.6μm filter (ULTA Prime PP 0.6μm from GE Healthcare Life Sciences, CA, USA), to retain cells and spores. 50ml samples were concentrated until 2ml using an Amicon Ultra-15 filter of 100KDa and CIB were stored at -20^oC.

Results and Discussion
Figure 1 shows that BtRS strain did not grow in C7: N1 medium, as was expected by the assays developed by Rivera in 1998. However, small differences in growing were obtained between C3.5:N4.5 and C1:N7 mediums (Fig. 1). Nonetheless, CIB size and concentration were greater in C3.5:N4.5 than in C1:N7, indicating carbon and nitrogen sources as well as its concentration are very important for crystal production and stability (Fig. 2a, b).

It is reported that crystal stability was maintained below pH 9 [1]; therefore, in the C3.5:N4.5 medium, ingredients and its concentrations were carefully selected to get pH values between 8 and 8.5 throughout the crystal production phase. In this sense, after 72 h BtRS strain initiated its growth in this medium the pH remained around 8.5 units, while in C1:N7 medium pH overpassed the 9.5 units. Therefore, CIB low production and its low stability found in the C1:N7 medium (Fig. 2b), must be directly correlated with nutrients (C1:N7) and pH values obtained, as a consequence of the high Nitrogen concentration utilized. Furthermore, extracellular proteases analysis developed from BtRS strain grown in C1:N7 medium shows its maximum enzyme activity between pH 9 to 10 (data not shown). In this sense, these enzymes could be carrying out soluble Cry1Ab protein degradation on the C1:N7 medium (Fig. 2b). On the other hand, Cry1Ab higher levels found in C3.5:N4.5 medium seems to be a consequence of the nutrients concentration, the lower pH and the higher crystal stability obtained (Fig. 2a, d).

Once CIB production was increased and its stability was enhanced with C3.5:N4.5 medium its purification was carried out using a simple filtration procedure, to improve the whole process. However, in this medium the BtRS strain produced bigger crystals than spores (Fig. 2a); therefore, to reduce crystal size the culture aeration was increased at 5:1 (1000/200ml) instead of the 5:3 (1000/600ml) that was used in the first assay [5].

Thus, BtRS strain was grown in 200 ml of C3.5:N4.5 medium at conditions mentioned above and obtained results show that CIB size decreased from ≥ 1.0 to ≤ 0.5µm (Fig. 2a, c, d). In this sense, crystal size reduction could be a consequence of the different replication rates between pHT315-1Ab plasmid and BtRS strain; because the cells number found in 5:1 proportion was higher with respect to those found in 5:3 (Fig. 1). In this sense, more bacterial cells found at the same time means faster replication rate and consequently less Cry1Ab protein production by cell.

The 0.85µm Bt spore size reported by Carrera and coworkers in 2007 [11] was not affected by using 5:1 proportion (Fig. 2a,c), therefore, a filtration procedure was carried out using a 0.6μm cut-off filter to separate CIB (≤ 0.5μm) from the spores (~0.85µm) (materials and methods). Crystal’s disaggregation was carried out at 37°C for 1h mixing 400μl of purified CIB with 100μl of 0.5M Na₂CO₃/NaHCO₃ pH 10.5 solution and toxin activation was carried out for 30 min at 37°C using the same reaction tube plus the addition of 20μg/ml of trypsin. PMSF at 1mM was used to stop the reaction and activated Cry1Ab toxin was analyzed by SDS-PAGE (Fig. 3). This figure shows both Cry1Ab tetrameric and monomeric bands of ~260KDa (lane 2) and ~65 KDa (lane 3), were visible at pH 8.5 and 10.5, respectively. Cry1Ab protein identification was corroborated by mass spectrometry analysis after bans were cut out from the gel (data not shown). The Cry1Ab activated protein was stored at -70°C until it was used in insect assays. Protein quantification was carried out using the Bradford methodology.

Cry1Ab toxicity was tested against Manduca sexta larvae, toxin was applied at 1, 0.5 and 0.05μg/cm² onto diet surface, incubated seven days at room temperature and insect survival was recorded. It is known that Cry1Ab toxin presents high specificity against Bt-R1 cadherin-like receptor from Manduca sexta [10]; in this sense, insecticidal assays developed in this work shown a LD₅₀ of 0.05µg/ml that corresponds with results previously reported[2]. Furthermore, these results corroborate that Cry1Ab protein produced in C3.5:N4.5 medium not suffered any modifications after all treatments [12,13].

Conclusion
CIB production was increased and its stability was enhanced using the C3.5:N4.5 medium with respect to C7:N1and C1:N7 frequently used mediums. CIB purification using a simple filtration procedure instead of gradient or solvents utilization seems to be an easier and more convenient alternative. For this reason, we believe this methodology is a friendly and non-expensive alternative to improve the production, stability and purification of Cry toxins.

Bibliography

1. Bravo, A., Likitvivatanavong, S., Gill, S. & Soberón, M. (2011). Bacillus thuringiensis: a story of a successful bioinsecticide. Insect. Biochem. Mol. Biol., 41(7), 423-431.
2. Rosas-García, N. (2009). Biopesticide production from Bacillus thuringiensis: an enviromentally friendly alternative. Recent. Pat. Biotechnol., 3(1), 28-36.
3. Griko, N. B., Rose-Young, L., Zhang, X., Carpenter, L., Candas, M., Ibrahim, M. A., Junker, M. & Bulla, M. A. (2007). Univalent binding of the Cry1Ab toxin of Bacillus thuringiensis to a conserved structural motif in the cadherin receptor BT-R1. Biochemistry, 46(35), 10001-10007.
4. Pitones, R. V., Bravo, A. & Olmos, S. J. (2017). Identification of a Bacillus thuringiensis surface layer protein with cytotoxic activity against MDA-MB-231 breast cancer cells. J. Microbiol. Biotechnol., 27, 36-42.
5. Keshavarzi, M., Salimi, H. & Mirzanamadi, F. (2005). Biochemical and physical requirements of Bacillus thuringiensis subsp. kurstaki for high biomass yield production. J. Agric. Sci. Technol., 7(1), 41-47.
6. Thomas, W. E. & Ellar, D. J. (1983). Bacillus thuringiensis var israelensis crystal deltaendotoxin: effects on insect and mammalian cells in vitro and in vivo. J. Cell Sci., 60, 181-197.
7. Rolle Roderick, L. (2013). An extensive characterization study of different Bacillus thuringiensis strains collected from the Nashville Tennessee area. Afr. J. Biotechnol. 12(30), 4827-4835.
8. Rivera, D. (1998). Growth kinetics of Bacillus thuringiensis batch, fed-batch and continuous bioreactor cultures. PhD Thesis, University of Western Ontario, Ontario, Canada.
9. Mendoza, G., Portillo, A., Arias, E., Ribas, R. M., Olmos, S. J., (2012). New combinations of cry genes from Bacillus thuringiensis strains isolated from northwestern Mexico. Int. Microbiol., 15(4), 209-216.
10. Carrera, M., Zandomeni, R. O., Fitzgibbon, J. & Sagripanti, J. L. (2007). Difference between the spore sizes of Bacillus anthracis and other Bacillus species. J. Applied. Microbiol., 102(2), 303-312.
11. Bravo, A., Gómez, I., Conde, J., Muñoz-Garay, C., Sánchez, J., Miranda, R., Zhuang, M., Gill, S. S. & Soberón, M. (2004). Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta., 1667(1), 38-46.
12. Olmos, S. J., Bañuelos, A. E. & Almanza, M. G. (2011). Insecticide Cry proteins of Bacillus thuringiensis with anti-cancer activity.
13. Jiménez-Juárez, N., Muñoz-Garay, C. & Gómez, I., et al. (2007). Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae. J Biol Chem., 282(29), 21222-21229.