Expression, Purification and Bioassay of Cry55Aa protein against tomato root knot Nematode, Meloidogyne incognita

 

A.              Manivannan1, K. K. Kumar3, S. Varanavasiappan3, S. Manimegalai1, K. Poornima2, B. C. Devrajan2, D. Sudhakar3, V. Balasubramani1

1Department of Agricultural Entomology, Tamil Nadu Agricultural University (TNAU), Coimbatore - 641003

2Department of Nematology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University (TNAU), Coimbatore - 641003

3Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University (TNAU), Coimbatore - 641003

*Corresponding Author E-mail: balasubramani.v@gmail.com

 

ABSTRACT:

A nematicidal Cry55Aa protein was expressed and purified from E. coli cells. Higher level of expression was observed in culture induced with 0.1mM of IPTG after four hours of induction. The Cry55Aa protein was purified by resuspending the crude protein in solubilizing buffer at pH 12 for 120 min. When Meloidogyne incognita was tested against different concentration of purified protein, a clear dose response lethality was observed with an LC50 value of 31.4g/ml.

 

KEYWORDS: Cry55Aa protein, expression, purification, bioassay, M. incognita.

 

 


INTRODUCTION:

Bacillus thuringiensis (Bt), a gram-positive soil bacterium, produces crystalline inclusions during sporulation, having insecticidal proteins called δ-endotoxins. Different Bt strains produce a large number of insecticidal crystal proteins, which are encoded by cry genes and these strains are used as bio-pesticides for several decades1. Crystal proteins are known to be toxic against different insect orders such as Lepidoptera (butterflies and moths), Coleoptera (beetles) and Diptera2, as well as nematodes3.

 

Cry proteins such as Cry5, Cry6, Cry12, Cry13, Cry14, Cry21 and Cry55 are reported as nematicidal4 and found to betoxic against several nematode genera including Criconemella, Globodera, Ditylenchus, Heterodera, Meloidiogyne, Helicotylenchus, Pratylenchus, Rotylenchus, Radopholus, Tylenchus and Bursaphalenchus.5 B. thuringiensis strains produce more than one type of toxic proteins (cry, vip, cytetc.) and to confirm the toxicity of a particular Cry protein, the respective cry gene is cloned and expressed in a heterologous bacteria like E. coli and used in bioassay studies6.

 

E. coli is one of the most attractive hosts7 for expression of heterologous protein with the advantages of fast growthin an inexpensive medium and the availability of a large number of cloning vectors8. In this study, an attempt was made to optimize heterologous protein expression in E. coli.

The plasmid pET29a containing cry55Aa gene under the control of a T7 promoter was used for heterologous expression of Cry55Aa protein. In order to increase the yield of the Cry55Aa protein, different concentrations of IPTGwere usedwith different duration of induction. Expressed Cry55Aa protein was purified by using solubilizing buffer with different pHlevels and incubation time. The purified Cry55Aa protein was tested against tomato root knot nematode, M. incognita.

 

MATERIALS AND METHODS:

Gene construct:

The recombinant E. coli strain BL21 harbouring pET29a-cry55Aa gene (Accession no: HG764207.1) which was originallycloned from indigenous Bacillus thuringiensis isolate, T44 of Bt collections of CPMB & B, TNAU.

 

Expression of Cry55Aa in E. coli BL21 cell:

Two E. colicultures viz. BL21 (DE3) harbouring vector pET29a with cry55Aainsert and BL21 carrying pET29a vector without insert were grown in LB broth (5ml) containing kanamycin 50mg/l overnight at 37oC. About 500l of overnight-grown culture was inoculated in 25 ml offresh LB broth with kanamycin 50mg/l and incubated at 37C in orbital shaker (180rpm) till OD600 value reached ~0.6. Then different concentration of IPTG viz., 0.01, 0.05, 0.10, 0.50 and 1.0mM was added to induce the protein expression and the cells were further grown at 37C for different durations (1, 2, 3, 4, 5 and 6 hrs) with a view to optimize the IPTG concentration andduration of induction. The cultures were harvested by centrifugation and washed with 1X TE buffer (10.0 mMTris-Cl, 1.0 mMEDTA, pH 8). Thepellet was dissolved in 5 ml of TE buffer containing 1 mM phenyl methyl sulfonyl fluoride and sonicated by using ultra-sonic liquid processor (Sonics and Material Inc., USA). Sonication was done with off pulsar mode for 4 min at 20amplitude (4x 1 min with a time interval of 1 min.). The broken cells were pelleted by centrifugation at 7000rpm for 15 min. The pellet was dissolved in the TE buffer and washed twice in the same buffer. The final product was suspended in 200l of sterile double distilled water containing 2mM PMSF and an aliquot of 5l was used for separation in SDS-PAGE.

 

Crystal protein purification

The crude Cry55Aa protein was collected by centrifugation and resuspended with solubilization buffer (8.7 mM tripotassiumcitrate, 43.4 mM citric acid and 10mM dithiothreitol) at different pH levels (pH 9.0, 10.0, 11.0 and 12.0) and incubated at 37oC for different durations (30, 60, 90 and 120 minutes) to solubilise theCry55Aa protein. The contents were spun out at 8,000 rpm for10min andprotein was precipitated from the supernatant by adding 1 M tripotassium citrate to adjust pH to 5.0 and incubated in ice overnight. The Cry55Aa protein was harvested by centrifugation for 10 min at 12,000rpm. The precipitated Cry55Aa protein was washed with ddH2O, dissolved in 20mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) (pH 8.0) and resolved in SDS-PAGE. The sds-page was performed as per the protocol given by Laemmli (1970).9

 

Lethal activity assay against M. incognita:

Initial M. incognita culture was collected from infested tomato fields from Coimbatore, Tamil Nadu and multiplied in potted tomato plants maintained in greenhouse condition at 25 2C. The J2 stage of M. incognita were hatched on a filter paper which was saturated with water at 20-25oC and used to test the toxicity of the Cry55Aa toxin and the assay was performed in 24 well plates. Each well with400l S medium10 contained purified Cry55Aa protein
at 10, 20, 30, 40 and 50g/ml, about 100J2 worms and 1 l of chloramphenicol (10mg/ml). BSA (20g/ml) was used as the control andeach treatment was replicated four times. After incubation of 5 days at 25C, 50l culture from each well wastransferred ontoglass slides and observation made on live and dead worms under a dissecting microscope. Observation were made on at least three samples of 50l from each well.

 

RESULTS AND DISCUSSION:

Optimization of protein expression by IPTG induction:

SDS-PAGE results showed that the protein expression increased gradually with increase in concentration of IPTG and maximum expression was observed with0.1mM IPTG (Plate1). The protein fromrecombinant E. coli BL21(harbouring pET29a+cry55Aagene) showed an expected prominent band of ~45 kDa on SDS-PAGE, which confirmed the cry55Aa gene expression, whereas no band was noticed in protein isolated from E. coli BL21 (harbouring pET29a vector alone).When IPTG (0.1mM) induction was done for different durations (1, 2, 3, 4, 5 and 6 hrs) to check the level of expression, higher level of expression of protein was observed in cultures after four hours of induction (Plate 2). The present findings are comparable with previous report11 suggesting that 0.1mM IPTG was enough to induce the protein expression.12 An IPTG concentration of 0.1mM and an induction period forfour hours were found to be optimum for Cry55Aa expression in E. coli BL21 cells. Conditions for recombinant protein expressionappears to be protein dependent13. For instance, induction with IPTG 10μM was appropriate to induce the cHSPA6 expression which was 100 times less than generally used concentration and 5 h of post-induction incubation period was found to be better to produce folded cHSPA614

 

 

 


 

Plate 1. Standardization of IPTG concentration for Cry55Aa protein expression (L- Protein ladder (10-315kDa), E-empty vector (pET29a), 1 to 5- pET29a with Cry55Aa induced with IPTG 0.01, 0.05, 0.10, 0.50 and 1.0Mm)

 

 

Plate 2. Standardization of induction period for Cry55Aa protein expression (L- Protein ladder (10-250kDa), E-empty vector (pET29a), 1 to 7- pET29a with Cry55Aa induced with 0.1mM IPTG for 0, 1, 2, 3, 4, 5 and 6 hrs)

 

 


Purification of Cry55Aa protein by alkali solubilization

Solubility of crystal protein was found to have significant effect on toxicity15. Proteolytic activation of protoxin, is a crucial step in Cry protein toxicity mechanism, andfound to be reliant on crystal protein dissolution in host intestine16. Some types of crystal protein from B. thuringiensiswere found to be soluble at pH 12, making it nontoxic to insect with gut pH of 9.017. However, when crystal protein arepresolubilized at high pH, these nontoxic crystal proteins exhibit significant toxicity to kill specific insect.

 

In this study, purification of Cry55Aa protein from recombinant E. coli (BL21) using solubilization buffer with different pH (9, 10, 11 and 12) and duration of induction (30,60,90 and 120 min) was attempted. Cry55Aa protein solubilization was maximum with pH 12, showing an expected prominentband of ~45 kDa on SDS-PAGE. When solubilization was done for different duration of incubation (30, 60, 90 and 120 minutes), solubilization was found to increase gradually from 30 to 120 minutes (Plate 3). The results are comparable with earlier reports on Cry5B (>pH10), Cyt (pH 9.5), Cry4B (pH 12)18, 19 and Cry6Aa2 (pH 9.5)20.

 


 

 

Plate 3. Purification of Cry55Aaprotein from recombinant E. coli (L- Protein ladder, P- Protein without solubilization; 1 to 4- in each gel represent 30, 60, 90 and 120 minutes of incubation at 37oC)

Table 1. Lethal activity assay of purified Cry55Aa protein against M. incognita

 

Number of nematodes

Per cent mortality

Per cent corrected mortality

LC50

Fiducial limit LC95

Treatment

Released

Dead

Lower

Upper

Cry55Aa protein (ug)

10

97.38

23.32

23.95 (29.29)c

14.01

 

 

 

 

 

31.4

 

 

 

 

 

162.66

 

 

 

 

 

1109.62

20

89.94

34.64

38.62 (38.40)b

30.61

30

87.93

41.98

47.76 (43.71)b

40.94

40

88.52

48.28

54.46 (51.46)a

48.51

50

87.25

55.50

63.57 (52.98)a

58.81

Control

91.96

10.64

11.55 (19.80)d

 

CD (P=0.5)

 

 

7.20

 

Mean of four replications. Values in parentheses are arcsine transformed. Means in a column followed by same superscripts are not significantly different at P≤0.05

 

 


Lethal activity of Cry55Aa protein on M. incognita:

The percent corrected mortality of Cry55Aa protein against M. incognita was 14.01, 30.61, 40.94, 48.51 and 58.81 at concentrations of 10, 20, 30, 40 and 50g/ml respectively (Table 1). Calculated LC50 value for Cry55Aa protein against second juvenile stage of M. incognita was 31.4g/ml (162.661109.62g/ml for 95% fiducial limits determined by profit analysis). The results are comparable with estimated LC50 values of Cry55Aa1 protein which was 23.2g/ml21 against M. haplaas. In addition, the combination of Cry6Aa and Cry55Aa was reported to have toxicity on M. incognita22. The LC50 value of crude protein from Btstrain YBT-021 against Tylenchorhynchus sp., M. hapla, Pratylenchusscribneri, Ditylenchus destructor and Aphelenchoides sp. was about 94.3g/ml, 35.62 g/ml, 75.65g/ml, 215.21g/ml and 128.76g/ml respectively23.

 

In the present study, have shown that the cry55Aa gene could be expressed in E. coli by inducing BL21-DE3 cells with 0.1 mM IPTG. The results presented here demonstrate that the Cry55Aa protein is toxic to M. incognita. This Cry55Aa is a potential nematicidal protein and could be included as a component of IPM, either as a formulated product or though transgenic means.

 

ACKNOWLEDGEMENT:

The financial support received from the Department of Biotechnology, Government of India (Project No. BT/PR7742/PBD/16/1035/2013) is gratefully acknowledged.

 

REFERENCES:

1.      Hofte, H. and Whiteley, H. R. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev., 53: 242-255.

2.      Sanahuja, G., Banakar, R., Twyman, R., Capell, M., and Christou, P. 2011. Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol. J. 9:283300.

3.      Wei, J. Z., Hale, K., Carta, L., Platzer, E., Wong, C., Fang, S. C. andAroian, R. V.2003. Bacillus thuringiensis crystal proteins that target nematodes. Proc Natl. Acad. Sci. USA. 100: 27602765.

4.      Crickmore, N. 2005. Using worms to better understand how Bacillus thuringiensis kills insects. Trends Microbiol., 13: 347350.

5.      Edowaazn, D. E., Pein, J. and Sooresu, J. J. 1989. Novel isolates of Bacillus thuringiensis having activity against nematodes. JP Patent 1067192. http://www.wikipatents.com/JP-Patent1067192.

6.      John, C.E. 2014. Escherichia coli as an Experimental Organism. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0002026.pub2

7.      Swartz, J.R. 2001. Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 12:195201.

8.      Jana, S. and Deb, J.K. 2005. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 67:289298.

9.      Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.

10.   Larry J. Bischof, Danielle L. Huffman, and Raffi V. Aroian. 2006. Assays for Toxicity Studies in C. elegans With Bt Crystal Proteins. Methods and Applications: K. pp 1139-154. Totowa, Humana Press Inc.

11.   Ariane, L. L., Jlia, F., Gabriela, S. E., Daniel, T. V., Fernanda, V. R., Mitermayer, G.R., Ricardo, G. and Marco, A.M. 2014. Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Research Notes7 (1): 671.

12.   Candan, Y., ˙Ilsen, Z., and Kirdar, B. 1998. Optimization of starting time and period of induction and inducer concentration in the production of the restriction enzyme EcoRI from Recombinant Escherichia coli 294. Turk. J. Chem. 22: 221 226.

13.   Sahdev, S., Khattar, S.K. and Saini, K.S. 2008. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem. 307:249264.

14.   Malik, A., Abdulrahman, M., Alsenaidy, Mohamed, E., Wajahatullah, K., Mohammed, S., Alanazi. and Mohammad, D. B. 2016. Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli. Saudi J Biol Sci. 23(3): 410419.

15.   Aronson, A.I., Han, E.S., McGaughey, W. and Johnson, D. 1991. The solubility of inclusion proteins from Bacillus thuringiensisis dependent upon protoxin composition and is a factor in toxicity to insects. Appl. Environ. Microbiol. 57:981986.

16.   Pardo, L.L., Muoz, G.C., Porta, H., Rodrguez, A.C. and Sobern, M.B. 2008. Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Peptides. 30:589595.

17.   Du, C., Martin, P.A. and Nickerson, K.W. 1994. Comparison of disulfide contents and solubility at alkaline pH of insecticidal and noninsecticidalBacillus thuringiensis protein crystals. Appl. Environ. Microbiol. 60:3847 3853

18.   Samir, N., Rumyana, B., Rumyana, K., Stefan, D., Ivan, M. and Ruud, A. M. 2008. Solubilization, activation, and insecticidal activity of Bacillus thuringiensis Serovar thompsoni HD542 crystal proteins. Appl. Environ.Microbiol.; 74(23): 71457151.

19.   Koller, C. N., Bauer, L. S. and Hollingworth, R.M.1992. Characterization of the pH-mediated solubility of Bacillus thuringiensis var. san diego native delta-endotoxin crystals. BiochemBiophys Res.Commun. 30;184(2):692-9.

20.   Hui, L., Jing, X., Qiaoni, Z., Liqiu, X. and Ziquan, Y. 2013. The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans. Appl. Microbiol. Biotechnol. 97:1013510142.

21.   Guo, S., Liu, M., Peng, D., Ji, S., Wang, P., Yu, Z. and Sun, M. 2008. New strategy for isolating novel nematicidal crystal protein genes from Bacillus thuringiensis strain YBT-1518. Appl. Environ. Microbiol., 74(22):6997-7001.

22.   Peng, D., Lujun, C., Fenshan, W., Fengjuan, Z., Lifang, R. and Ming, S. 2011. Synergistic activity between Bacillus thuringiensis Cry6Aa and Cry55Aa toxins against MeloidogyneIncognita. Microbial Biotechnology. 4(6), 794798.

23.   Yu, Z.Q., Wang, Q.L., Liu, B. and Zou, X. 2008.Bacillus thuringiensis crystal protein toxicity against plant-parasitic nematodes. Chinese journal of Agricultural Biotechnology. 5(1):11-17.

 

 

 

 

 

Received on 11.11.2019 Modified on 31.12.2019

Accepted on 21.01.2020 A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2020; 12(1):. 19-23.

DOI: 10.5958/0975-4385.2020.00004.7