Biotechnol Lett (2011) 33:531–537 DOI 10.1007/s10529-010-0452-0

ORIGINAL RESEARCH PAPER

Identification of the minimal active fragment of the Cry1Ah toxin Jing Xue • Zishan Zhou • Fuping Song • Changlong Shu • Dafang Huang • Jie Zhang

Received: 28 September 2010 / Accepted: 22 October 2010 / Published online: 3 November 2010 Ó Springer Science+Business Media B.V. 2010

Abstract cry1Ah1, a novel holo-type gene cloned from Bacillus thuringiensis strain BT-8, encoded a protein exhibiting strong insecticidal activity against lepidopteran insects. To identify the minimal active fragment of the Cry1Ah toxin, 9 pairs of primers were designed to generate different PCR products. Seven PCR products were amplified by different primers using the cry1Ah1 gene as a template and cloned into a pET-21b vector. These positive clones were separately transformed into Escherichia coli. Insecticidal activity against 2nd-instar larvae of Plutella xylostella was performed using the leaf-dip bioassay: the minimal active fragment of the Cry1Ah toxin was located between amino acid residues 50I and 639E.

Electronic supplementary material The online version of this article (doi:10.1007/s10529-010-0452-0) contains supplementary material, which is available to authorized users. J. Xue  Z. Zhou  F. Song  C. Shu  J. Zhang (&) State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China e-mail: [email protected] D. Huang Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China

Keywords Bacillus thuringiensis  Cry1Ah  Toxicity  Minimal active fragment  Plutella xylostella

Introduction Insecticidal crystal proteins (ICPs) are a group of proteins that form crystalline inclusions produced by Bacillus thuringiensis during sporulation (Schnepf et al. 1998). ICPs exhibit specific toxicity against lepidopteran, dipteran, coleopteran and hymenopteran insects, as well as other types of pests including nematodes and mites. Since the first cry gene was cloned from B. thuringiensis ssp. kurstaki HD-1 in 1981 (Schnepf and Whiteley 1981), 560 cry genes have been characterized (http://www.lifesci.sussex. ac.uk/home/Neil_Crickmore/Bt/toxins2.html). The discovery of these genes has led to the rapid development of transgenic crops with insecticidal traits (Schnepf et al. 1998). Genetically-engineered (GE) plant varieties expressing Cry proteins from B. thuringiensis are the most widely grown insect-resistant transgenic plants to date. Furthermore, the importance of transgenic plants expressing novel Cry proteins will likely increase in coming years. Most ICPs are synthesized in the form of crystalline protoxins. These protoxins are solubilized in the alkaline midgut of susceptible larvae and activated by trypsin-like gut proteases to produce a mature toxin, leading to insect death (Schnepf et al. 1998). As only

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the N-terminal fragments of the Cry proteins play an important role in the insect death, we only need transform the truncated forms of cry gene into plant to get a high expression level of heterologous protein. As previous studies have shown, the toxicity and expression level of full-length cry genes in transgenic plants vary significantly from those of parental strains. The expression levels of Cry1A gene, in addition to their toxicity against targets insects, were substantially decreased when the full-length gene was expressed in transgenic tobacco (Koziel et al. 1993). However, the expression levels of the truncate fragment of Bt2 insecticidal crystal protein were much higher than those of full-length proteins in transgenic tobacco (Vaeck et al. 1987). In contrast, the active region of Cry1Ie1 is 648 amino acid residues away from N-terminus (Wu et al. 2003), of which a high level of expression has previously been induced in tobacco (Liu et al. 2004). Therefore, it is critically important to confirm the minimally active fragment of the cry gene before transfecting the gene into plants. The cry1Ah gene, cloned from B. thuringiensis BT-8 strain, yields a protein that is highly toxic against Helicoveper armigera, Chilo suppressalis and Ostrinia furnacalis. The protoxin of Cry1Ah is 134 kDa and, after being subjected to trypsin digestion, an active Cry1Ah toxin 65 kDa is produced (Xue et al. 2008). The cry1Ah1 gene was transformed into corn (Wang et al. 2008) though the toxicity of the protein was not as high as predicted; therefore, it is necessary to determine the minimal active fragment of the cry1Ah gene to increase its toxicity in the crop. To reveal the minimal active fragment, several truncated Table 1 DNA fragments, and corresponding polypeptides, amplified using different primer pairs

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genes were amplified, the proteins were expressed in Escherichia coli and the bioassays were conducted.

Materials and methods Bacterial strains, plasmids and growth conditions E. coli strain Rosetta2 (DE3) (http://www.merckchemicals.com.cn) was used to express Cry proteins. The pET-21b plasmid was used as an expression vector. All E. coli and B. thuringiensis strains were grown in Luria–Bertani (LB) medium with shaking at 230 rpm. Ampicillin was added to autoclaved media at 100 mg/ml for E. coli strains JM110 and Rosetta2 (DE3). Gene analysis and primer design Based on the cry1Ah1 nucleotide sequence (GenBank accession number: AF281866) that was obtained in our previous study (Xue et al. 2008), the cry1Ah1 sequence and its deduced amino acid composition were analyzed using the NCBI BLAST sequence analysis tool; three conserved amino acid domains and corresponding conserved nucleotide sequences were identified. Based on the results of the BLAST analysis, several primers were designed to amplify the different fragments of the cry1Ah1 gene (listed in Table 1). A pair of DNA primers designed to amplify the full-length of the cry1Ah gene were synthesized at the same time. For vector construction, BamHI and SalI sites were added to the 50 end of the primers.

Primer pair

Nucleic acid site

Amino acid site

DNA length (bp)

AA length

Polypeptide

HF/HR

1–3546

1–1182

3546

1182

HF/PR2001

1–2001

1–667

2001

667

Cry1Ah1-a

PF148/PR1971 PF325/PR1971

148–1971 325–1971

50–657 109–657

1824 1647

608 549

Cry1Ah1-b Cry1Ah1-c

HF/PR1635

1–1635

1–545

1635

545

Cry1Ah1-d

HF/PR1863

1–1863

1–621

1863

621

Cry1Ah1-e

PF58/PR1917

58–1917

20–639

1860

620

Cry1Ah1-f

PF58/PR1920

58–1920

20–640

1863

621

Cry1Ah1-g

PF148/PR1917

148–1917

50–639

1770

590

Cry1Ah1-h

PF148/PR1920

148–1920

50–640

1773

591

Cry1Ah1-i

Cry1Ah1

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Gene amplification and the construction of expression plasmids pSXY422-1Ah (pSXY422 vector carrying the fulllength cry1Ah1 gene, Xue et al. 2008) was used as PCR template. Truncated cry1Ah genes were obtained by PCR amplification using different primer pairs. The PCR products were digested using BamHI and SalI restriction enzymes, and the resulting truncated cry1Ah genes were cloned into the pET21b vector between the BamHI and SalI sites. The integrity of the recombinant plasmid DNA was confirmed by restriction analysis and sequencing. The positive clones were named between pET1Ah-a to pET1Ah-i, as shown in Table 1. All DNA manipulations, including restriction digestion, ligation, agarose gel electrophoresis and transformation, were performed by standard protocols. The expression of truncated Cry1Ah proteins in E. coli To express the truncated Cry1Ah toxin, these clones were transformed into the E. coli Rosetta2 (DE3) strain. E. coli containing the various cry1Ah genes were grown in 1 l LB medium to an OD600 of 0.5; expression of the transformed genes was then induced with 0.5 mg IPTG/ml. After 8 h at 37°C, the cells were pelleted, resuspended in 50 ml of 20 mM Tris/ HCl (pH 8.0) buffer and sonicated for 5 min, then centrifuged at 10,0009g for 10 min at 4°C. Supernatants were collected and the pellets resuspended in 20 mM Tris/HCl (pH 8.0) buffer. Protein was analyzed by SDS-PAGE (8%), with BSA as a standard. Insect bioassays Insecticidal activities of the truncated Cry1Ah against larvae of the diamondback moth (P. xylostella) were evaluated. The bioassay procedure was conducted on fresh cabbage leaves using a leaf-dip bioassay (Tabashnik et al. 1993). The truncated Cry1Ah proteins were serially diluted in 20 mM Tris/HCl (pH 8.0) buffer. Biot1Ah (HD73- strain transformed with pSXY422-1Ah vector containing the cry1Ah1 gene) was used as the control. Cabbage leaves were dipped in 30 ml aliquots of either toxin or 20 mM Tris/HCl (pH 8.0) buffer (as a control); twenty 2nd-

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instar larvae were then placed on prepared leaf disks. After 4 days, the number of surviving larvae was recorded. All bioassays were repeated three times, and the median lethal concentration (LC50) values were calculated using probit analysis (Finney 1971).

Results Sequence analysis Sequence analysis using the NCBI BLAST sequence analysis tool revealed that Cry1Ah contained three conserved domains (Fig. 1). Compared with Cry1Ac, we deduced that the two proteins had the similar activity sites (Fig. 1). Construction of recombinant plasmids and analysis of recombinant protein synthesis The full-length and several truncated forms of the cry1Ah1 gene were cloned into the pET-21b plasmid and sequenced. The recombinant plasmids were transformed into E. coli strain Rosetta2 (DE3). Protein expression was analyzed by SDS-PAGE. The size of full-length Cry1Ah1 protein was approx. 130 kDa, as seen in Lane 1 (Fig. 2). Cry1Ah1-a was the largest of the nine truncated proteins (Lane 3), with molecular masses of roughly 73 kDa; Cry1Ah1-c (Lane 9) and Cry1Ah1-d (Lane 11) were the smallest, with molecular masses of roughly 60 kDa. Cry1Ah1-b (Lane 5) had a molecular mass of roughly 66 kDa; Cry1Ah1-e (Lane 13) and Cry1Ah1-g (Lane 19) were slightly larger, both with molecular masses of roughly 68 kDa. Cry1Ah1-h (Lane 21) and Cry1Ah1-i (Lane 25) were slightly smaller, both with the molecular masses of roughly 65 kDa (Fig. 2). Bioassays The LC50 values of the different polypeptides are listed in Table 2. The truncated proteins Cry1Ah1-c, Cry1Ah1-d and Cry1Ah1-e were not toxic against the larvae. However, there was no substantial difference between the other truncated proteins and the fulllength Cry1Ah expressed in E. coli. These results revealed that the active fragment of the Cry1Ah toxin was located between amino acid residues 50I and 639E.

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Biotechnol Lett (2011) 33:531–537 Cry1Ac1·····························································MDNNPN I NEC I PYNCLSNP·····19 Cry1Ah 1 MKNS I KLSELWYFNERKWRYFME I VNNQNQCVPYNCLNNP·····40 Cry1Ac1·····EVEVLGGERI ETGYTP I D I SLSLTQFLLSEFVPGAGFVLG·········59 Cry1Ah1······EI EI LEGGRI SVGNTP I D I SLSLTQFLLSEFVPGAGFVLG·········80 49

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Cry1Ac1·····VLDI VALFPNYDSRRYPI RTV SQLTREI YTNPVLENFDGS·········279 Cry1Ah1·····VLDI VALFPNYDSRRYPI RTVSQLTREI YTNPVLENFDGS·········300 275

280

Cry1Ac1······FSNSSVS I I RAPMFSWI HRSAEFNNI I ASDS ITQI PAVKG········479 Cry1Ah1·····SS · SSVS I I RAPMFSWI HRSAEFNNI I ASDSI TQI PAVKG········499 491

481

Cry1Ac1·····TSSLGN I VGVRNFSGTAGV I I DRFEF I PVTATLEAEYNLE······619 Cry1Ah1·····TSSLGN I VGVRNFSGTAGV I I DRFEF I PVTATLEAEYNLE······639 629

Cry1Ac1·····RAQKAVNALFTSTNQLGLKTNVTDYH I D·····································647 Cry1Ah1·····RAQKAVNALFTSTNQLGLKTNVTDYH I D·····································667 640

Fig. 1 Sequence analysis of Cry1Ah and Cry1Ac proteins. The sequences of the cry1Ah1 gene and its deduced amino acid residues were analyzed using the NCBI BLAST sequence analysis tool. Three conserved sequences were identified: Domain I: amino acid residues 57–275; Domain II: amino acid pET1Ah1 kDa

H

I

pET1Ah1-a pET1Ah1-b

S

I

S

I

residues 280–481; Domain III: amino acid residues 491–629. The identity between Cry1Ah and Cry1Ac is 86%. Similar to Cry1Ac, Cry1Ah contains tryptic digest sites at amino acid residues 49R and 640R

pET1Ah1-c pET1Ah1-d pET1Ah1-e

pET21b

S

I

S

H

I

S

I

S

I

S

pET21b

I

S

212 116 97 66 45

pET1Ah1-f pET1Ah1-g pET1Ah1-h kDa

H

I

S

I

S

I

S

pET21b

I

S

pET1Ah1-i

H

I

S

pET21b

I

S

212 116 97 66

45

Fig. 2 SDS-PAGE analysis of the Cry1Ah1 polypeptide expressed in E. coli. H: protein standard (high range). I resuspended pellet; S collected supernatant

Discussion Nine truncated cry1Ah1 genes were successfully expressed in E. coli, according to the results of our

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SDS-PAGE analysis. Although the full-length cry1Ah1 gene was expressed in the same form as in B. thuringiensis (i.e., with a molecular mass of approx. 130 kDa), the toxicity of the E. coli-

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Table 2 Toxicities of truncated Cry1Ah1 proteins against P. xylostella larvae Protein

Location (AA) LC50 (lg/ml) 95% Limit

Cry1Ah1 (Bt)

1–1182

1.38

0.87–1.98

Cry1Ah1 (E. coli) 1–1182

6.56

4.58–8.44

Cry1Ah1-a

1–667

13.03

9.33–18.15

Cry1Ah1-b

50–657

11.52

4.64–17.70

Cry1Ah1-c

109–657

NA

Cry1Ah1-d

1–545

NA

Cry1Ah1-e

1–621

NA

Cry1Ah1-f

20–639

9.34

2.11–17.00

Cry1Ah1-g

20–640

12.26

8.76–16.38

Cry1Ah1-h

50–639

7.70

1.74–13.35

Cry1Ah1-i

50–-640

8.31

5.71–10.34

NA no activity

expressed protein was approx. 75% less than that expressed in B. thuringiensis strain (Table 2). This might because the protein expressed in E. coli was insoluble or because the insoluble protein may be absorbed differently. Cry1 protoxins of B. thuringiensis are insecticidal proteins approx. 130–140 kDa. After being ingested into the alkaline environments of the insect midgut, activated toxins with molecular masses of approx. 60–65 kDa are released. According to the pore formation model, the activated toxins bind to primary receptors (e.g., cadherin-like proteins), facilitating further protease cleavages of the N-terminal ends of the toxins and eliminating the helix a-1 of Domain I (Go´mez et al. 2002). This cleavage induces the assembly of oligomeric forms of the toxin. The oligomers exhibit increased binding affinity to secondary receptors (such as APN) (Jurat-Fuentes and Adang 2004) and then insert into the apical membranes of midgut cells. This results in the formation

of pores causing osmotic shock and bursting of the midgut cells, finally lead to the death of insect (Sobero´n et al. 2008). The Cry1A protein is digested into its activated form at amino acid residue 28R of N-terminus and residue 623K of the C-terminus (Nagamatsu et al. 1984; Bietlot et al. 1989). It is well known that Trypsin and chymotrypsin are the primary proteases in the midgut; on the basis of sequence analysis and the location of arginine and lysine residues, the insecticidal activities of 10 different Cry1Ah toxin fragments were investigated: a truncated protein consisting of amino acid residues 50I to 639E exhibited the same toxicity as the full-length Cry1Ah protein. This truncated protein was similar to the 28R-619E fragment of Cry1Ac. Truncation of 108 amino acids from N-terminus resulted in a complete loss of toxicity against P. xylostella. According to the structural prediction, Domain I of Cry1Ah falls between amino acid residues 57I and 275R (Fig. 1), and the deletion of 108 amino acids from the N-terminus results in the loss of 51 amino acids from Domain I (corresponding to helices a-1 to a-3) (shown in yellow in Fig. 3a). Previous studies showed that Domain I was a bundle of seven a-helices in which the central helix (a-5) was hydrophobic and encircled by six other amphipathic helices; this helical domain is responsible for membrane insertion and pore formation (Grochulski et al. 1995). Therefore, altering the structure of Domain I may positively modulate the correct insertion of the protein into the cell membrane or pore-formation ability. However, modified Cry1Ab and Cry1Ac toxins that lack helix a-1 can form oligomers without cadherin and can kill insects that show greatly decreased susceptibility to native Cry1A toxins due to RNAi silencing or mutations in the

Fig. 3 The 3-D structure of Cry1Ah Domain I and Domain III. a Domain I (residues 57–275), the yellow region represents amino acid residues 57I to 108R. b Domain III (residues 491–629), the yellow region represents amino acid residues 621R to 629A

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cadherin gene (Sobero´n et al. 2007). This means that the absence of helix a-1 has no effect on the toxicity or the insertion of the protein into cell membranes. Therefore, we can conclude that helices a-2 and a-3 are necessary for correct oligomer formation and the lack of this region may influence the ability of the protein to form pores and result in a loss of toxicity. In contrast, we can predict that Cry1Ah is digested at amino acid residue 49R of the N-terminus like other Cry1A proteins (Cry1Ac is digested at residue 28R) because neither arginine nor lysine exists in the region from residues 50I to 107Q. Although the truncated Cry1Ah toxin retained its toxicity against P. xylostella after the amino acid residues 657L to 639E on the C-terminus were deleted, it completely lost toxicity against P. xylostella when it was truncated at residue 621D of the C-terminus. A previous study showed that Domain III was involved in receptor binding (Lee et al. 1995; Bravo et al. 2005). As predicted, Cry1Ah Domain III ranged from amino acid residues 491I to 629T (Fig. 1) and truncation at residue 621D resulted in the deletion of nine amino acids of Domain III (part of the b-23 region) (shown in yellow color in Fig. 3b). According to homologous analysis of the sequences, there are five highly conserved blocks in most Cry proteins; these are located either at the center of three domains or the linker regions between the domains and play a key role in determining the structure of the Cry proteins. The fifth conservative block of Cry1Ah is located in the center of Domain III (corresponding to b-23); therefore, the deletion of 9 amino acids in the sheet b-23 destroys the structure and function of Domain III and results in a loss of toxicity. According to our previous study, the deletion of sheet b-23 resulted in a loss of toxicity of Cry1Ba3 against P. xylostella (Wang et al. 2005). Furthermore, deletion of the same domain in the Cry1Ie1 proteins resulted in a complete loss of toxicity against P. xylostella and O. furnacalis (Wu et al. 2003). Moreover, amino acid residue 639E in Cry1Ah is the same site as residue 619E in Cry1Ac; previous studies have shown that Cry1Ac is digested at residue 623K, suggesting that the truncated form of Cry1Ah polypeptide lacks 4 amino acids relative to activated Cry1Ac but retains the toxicity of the fulllength protein. Therefore, we predict that the digestion site is 640R on Cry1Ah.

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In summary, the active fragment of the Cry1Ah lies between amino acid residues 50I and 639E, a-2 and a-3 helices of Domain I and b-32 sheet of Domain III are important for its toxicity against P. xylostella. We further conclude that when attempting to minimize the active fragment of Cry1Ah in future studies, it is best to truncate amino acids oneby-one. Acknowledgments This study was support by the 973 Projects of China (2009CB118902 and 2007CB109203) and National S&T Major project (2008ZX08001-001). We thank Ms. Yingping Liang for insect rearing and bioassays.

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