Construction of a synthetic gene encoding the red scorpion neurotoxin ButaIT and assembly of expression constructs

A synthetic gene containing the entire coding sequence for the mature ButaIT polypeptide (as given in Genbank [AF481881]) was assembled from overlapping oligonucleotides. Each of the two complementary strands of the gene contained four 30-mers and one 15-mer, arranged so that overlaps of 15 bases between complementary portions of the oligos were present. The oligonucleotides were phosphorylated prior to annealing, and the desired product was amplified by PCR after annealing and ligating, using the 15-mer oligonucleotides at the 5' end of each strand as primers. A DNA fragment corresponding to the correct size for the complete product was obtained, and was excised from gel, cloned in an intermediate vector, and verified by DNA sequencing. The resulting fragment corresponded to the desired coding sequence, with additional restriction sites introduced at the ends to allow for subsequent cloning.

The ButaIT coding sequence was assembled into two constructs for expression of proteins in the yeast Pichia pastoris using the shuttle expression vector pGAPZαB. The first construct contained ButaIT alone, and the second encoded a fusion protein in which ButaIT is fused to the N-terminus of residues 1 – 105 of mature GNA via a 3 amino acid linker peptide (Fig. 1). Both constructs were arranged so that the recombinant proteins were produced as precursors containing the yeast α-factor prepro-sequence at the N-terminus; this sequence directs the product into the secretory pathway in the expression host, and is removed during translation and subsequent transport. The final protein product for ButaIT is predicted to contain a 9 amino acid C-terminal extension, containing the (his)₆ tag, and is predicted to have 2 extra alanine residues at the N-terminus after removal of the prepro- sequence, The predicted protein product for ButaIT/GNA contains a C-terminal extension of 23 amino acids, including a myc epitope tag and the (his)₆ tag (Fig. 1). Constructs were assembled by restriction/ligation and cloning in E. coli, and were verified by DNA sequencing.

Expression and purification of recombinant ButaIT and ButaIT/GNA

DNA from verified clones of the expression construct plasmids was linearised and transformed into competent cells of a protease-deficient Pichia pastoris strain. Since the expression vector contains a constitutive yeast promoter, clones selected as positive for transformation by antibiotic resistance and colony PCR were tested for expression of recombinant proteins in small-scale cultures. Culture supernatants were assayed for the presence of recombinant proteins by immuno-dot blot and Western blotting, using antibodies to the C-terminal tags, or in the case of the ButaIT/GNA fusion, to GNA. Clones expressing recombinant proteins at higher levels (relative to other clones) were selected for further study.

For production of recombinant proteins, selected yeast clones expressing ButaIT and ButaIT/GNA were grown in a bench-top fermenter. ButaIT and ButaIT/GNA were partially purified from culture supernatant by hydrophobic interaction chromatography on phenyl-Sepharose. Recombinant ButaIT was produced in relatively low yield (<1 mg/l culture). The partially purified material contained a polypeptide of estimated mol. wt. approx. 10,000 as the major component (fig. 2A); when a gel separation was analysed by Western blotting, and probing with with anti-(his)₆ antibodies, the band gave a strong and specific reaction, showing that this band represents recombinant ButaIT. The discrepancy in size between predicted and estimated mol. wts. for this polypeptide is consistent with previous results in which small toxin polypeptides with high cysteine content show anomalous migration on SDS-PAGE.

ButaIT/GNA was produced at a yield of 25–35 mg/l culture medium, and was the major protein component of the material eluted from the hydrophobic interaction column by a water wash. Further purification of ButaIT/GNA by gel filtration was carried out to remove contaminating yeast proteins (fig. 2B). Analysis of purified ButaIT/GNA by SDS-PAGE (Fig. 2C) shows one major component of approx. 18.5 kDa, which is close to the predicted molecular weight of 19.4 kDa. A minor component (weakly stained band) of approx. 17 kDa is also present. Both bands were immunoreactive towards anti-GNA antibodies (data not presented), but only the major component reacted with anti-(his)₆ antibodies. The indicated size of these polypeptides, and their immunoreactivity, suggest that the majority of ButaIT/GNA is full-length protein with the C-terminal extension intact, with a small proportion of ButaIT/GNA from which the C-terminal his tag has been removed. Recombinant ButaIT/GNA agglutinated rabbit erythrocytes at a comparable concentration to native GNA (50 μg/ml for ButaIT/GNA, vs. approx. 10 μg/ml for GNA), showing that the lectin part of the fusion protein is functional.

While the ButaIT/GNA could be produced as intact fusion protein, prolonged exposure to proteases, such as those in yeast culture supernatant, resulted in varying amounts (normally <50%, as estimated by band staining intensity) of proteolytic cleavage. Proteolysis occurred predominantly at or near the linker region between ButaIT and GNA, and generated a polypeptide, mol. wt. approx. 12,000, which reacted with anti-GNA and anti-(his)₆ antibodies. When band intensities were compared for the same protein samples between stained gels and Western blots, it was apparent that the anti-GNA antibody gave a stronger reaction with the 12,000 mol. wt band than with the intact fusion protein; as a consequence, the extent of cleavage of the fusion is overestimated in Western blots.

Toxicity of recombinant proteins to tomato moth larvae after injection into the haemocoel

The toxicity of recombinant ButaIT was demonstrated by injection into fifth stadium L. oleracea larvae (approx. 30 – 60 mg). Injection of the partially purified recombinant protein at doses of the order of 1–10 μg caused a decline in larval suvival over a period of 7 days, to less than 30%. Control survival (buffer-injected, or injected with other partially purified recombinant proteins) was >70% over 7 days. The effect was dose-dependent, and at high doses of ButaIT 100% mortality was observed after 3 days, during which time control survival was >90%. These results could not be compared with previously published data for toxicity of the protein purified from scorpion venom, as the recombinant protein could not be quantified, and attempts to purify it further were not successful. However, the data show that expression as a recombinant protein in P. pastoris did not abolish the toxicity of ButaIT.

The ButaIT/GNA fusion protein was also toxic when injected into 5^th stadium L. oleracea larvae. The major effect on mortality was seen during the first two days after injection, after which survival (particularly at higher doses) stabilised. The effect was dose dependent; injection of 20 μg of fusion protein per larva (approx. 500 μg per g insect; n = 10) reduced survival to 40% after 6 days, whereas injection of 4 μg of fusion per larva (approx. 100 μg per g insect; n = 25) reduced survival to 60%. Control survival was 97% over this interval (n = 40). Injection of GNA at 20 μg per larva (approx. 500 μg per g insect; n = 20) had no effect on survival, which was 95% over 6 days (fig. 3); thus the toxic effect shown by ButaIT/GNA on injection must be due to the toxin fused to GNA. Surviving insects that had been injected with ButaIT/GNA also showed a reduction in weight gain compared to controls (PBS-injected). The effect was variable, but in a representative assay injection of approx. 100 μg per g insect of the fusion protein reduced weight gain over 6 days by approx. 40% (starting weight 38 ± 2 mg, final weight for controls 205 ± 17 mg, for ButaIT/GNA injected insects 134 ± 19 mg, n = 20 for each treatment, significantly different at p < 0.01). Injection of GNA at similar dose had no effect on weight gain.

Toxicity of recombinant proteins to tomato moth larvae after oral delivery via artificial diet

Third stadium tomato moth larvae were exposed to diet containing recombinant ButaIT/GNA at 2.5% and 4.5% of dietary protein. Delivery of the toxin via this route had little or no acute toxicity to larvae, but long-term effects on larval survival and development were observed. As shown in Fig 4A, survival of larvae fed on diet containing ButaIT/GNA at 4.5% of total protein declined significantly after day 8 of the assay, so that only 75% of larvae remained alive after 12 days of feeding, compared to 100% of larvae fed on control diet. Exposure to the lower dose of ButaIT/GNA did not have a significant effect on survival over a similar time period. Similarly, exposure to GNA in the diet at 5% of total protein had no effect on survival over 12 days.

Surviving larvae fed on the ButaIT/GNA-containing diets showed a significant reduction in mean larval weight compared to the control diet treatment for all time points after day 4 of the bioassay (ANOVA; p < 0.05), so that by day 12 of the bioassay the mean weight of surviving ButaIT/GNA-fed larvae (both doses) was approx. 40 % less than that recorded for control insects (Fig. 4B). The consumption of diet correlated with larval growth, so that overall, ButaIT/GNA at 4.5 % of total protein caused a reduction of approx. 35 % of total diet consumed, compared to control treatment, and ButaIT/GNA at 2.5 % of total protein caused a reduction of approx. 30% in diet consumption. Feeding GNA at 5% of total protein in the diet had only marginal, insignificant effects on growth and consumption (Fig. 4B).

Presence of ButaIT/GNA in tomato moth larval tissues after oral administration

Gut tissue and haemolymph samples were extracted from larvae exposed to ButaIT/GNA at 2.5 % of dietary protein for 24 h and 8 days, and from larvae exposed ButaIT/GNA at 4.5 % of dietary protein for 12 days. Typically samples were pooled from 3 insects to give 2 – 4 replicates in each instance. Western blot analysis, using anti-GNA antibodies as a probe, was used to verify binding of the fusion protein to the guts of orally exposed larvae, and uptake of ButaIT/GNA into the haemolymph. Fig. 5 represents a summary of the results obtained. In all cases immunoreactivity of two major bands with anti-GNA antibodies was observed in samples from insects fed ButaIT/GNA, but not control insects. These bands, present in both gut and haemolymph samples, correspond in molecular weight to the sizes of ButaIT/GNA and GNA. The susceptibility of fusion proteins based on GNA to proteolytic cleavage in the region of the linker peptide between GNA and the fused peptide/polypeptide has been observed previously for other recombinant fusion proteins [11, 12], and has been noted above for ButaIT/GNA. When compared to standards, the ButaIT/GNA fusion protein detected in both gut and blood samples from ButaIT/GNA-fed larvae showed a reduction in the intensity of the band corresponding to intact fusion protein, and a concurrent increase in the band corresponding to GNA. The results implied that limited proteolysis of the ButaIT/GNA fusion occurs during ingestion, passage through the gut, and transport to the haemolymph. Analysis of gut contents from ButaIT/GNA-fed insects by Western blotting suggested that most of the proteolytic cleavage of the fusion protein occurred during the digestive processes occurring in the gut lumen (fig. 5). Analysis of proteins extracted from artificial diet showed that the fusion protein was not proteolytically cleaved as a result of incorporation into the diet, and that it remained intact over a period of 2–3 days (Fig. 5).

Effects of ButaIT/GNA on non-lepidopteran insects

The toxicity of ButaIT/GNA towards non-lepidopteran insects was tested using the homopteran species Nilaparvata lugens (rice brown planthopper). The fusion protein was fed to insects by incorporation into liquid diet at a concentration of 0.05% (0.5 mg/ml diet), and its effects on survival were compared to GNA at a similar concentration. Results are shown in fig. 6. Control survival over the 8-day bioassay was approx. 40%, and at day 6, when all insects on the "water only" control had died, was approx. 50%. Corrected mortality values, based on day 6 of the assay, were 64 ± 11% for GNA and 92 ± 5% for ButaIT/GNA; these are significantly different at p < 0.01 (survival analysis). At all times after day 1 the effect of ButaIT on survival was greater than that of GNA in these bioassays. Western blotting analysis of samples of diet after N. lugens feeding showed that ButaIT/GNA was stable in the diet over a 2-day interval, with only a small amount of proteolysis of the fusion observed (data not presented).

Insects

Lacanobia oleracea were reared continuously on artificial diet (Bown et al., 1997) at 25°C under a 16 h:8 h light:dark regime. Nilaparvata lugens were kept on 20 day old rice plants (Oryza sativa variety 'TN1'), at 28°C, 90% RH, under a 16 h:8 h light:dark regime.

Materials and recombinant DNA techniques

A cDNA containing the GNA coding sequence has been described previously [15]. Sub-cloning was carried out using the TOPO cloning kit (pCR2.1 TOPO vector) purchased from Invitrogen [16]. P. pastoris X33 strain and SMD1168H (Protease A deficient) strain, the expression vector pGAPZαB, and Easycomp Pichia transformation kit were also from Invitrogen. Oligonucleotide primers were synthesised by Sigma-Genosys Ltd. [17]. Restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, and Pfu DNA polymerase, were supplied by Promega [18]. Plasmid DNA was prepared using Promega Wizard miniprep kits. GNA was obtained from Vector Laboratories Inc. or was produced as a recombinant protein in yeast [13]. Anti-GNA antibodies, raised in rabbits, were prepared by Genosys Biotechnologies, Cambridge, UK, and anti-(His)₆ (C-terminal) antibodies were from Invitrogen.

General molecular biology protocols were as described by Sambrook and Russell [19] except where otherwise noted. All DNA sequencing was carried out using dideoxynucleotide chain termination protocols on Applied Biosystems automated DNA sequencers by the DNA Sequencing Service, School of Biological and Biomedical Sciences, University of Durham, UK. Sequences were checked and assembled using Sequencher software [20] running on Mac OS computers.

Assembly of expression constructs for recombinant proteins

The ButaIT amino acid sequence (Genbank [AF481881]) was used as the basis for the assembly of a synthetic ButaIT gene. Each strand (i.e. coding and complementary strands) of the sequence encoding the mature ButaIT chain (114 nucleotides) was subdivided into 5 fragments, in such a way that each fragment overlapped neighbouring fragments on the complementary strand by 10–15 bases. Ten oligonucleotide primers based on these fragments were synthesised, and used in an assembly reaction of the full mature ButaIT coding sequence.

The oligonucleotides used were as follows:

Coding strand:

5'-TCGCCTGCAGCAAGGT-3'

5'-GTGGTCCTTGCTTTACAACTGATCCTCAAA-3'

5'-CACAAGCCAAGTGTAGTGAGTGTTGTGGGC-3'

5'-GAAAGGGTGGAGTATGCAAGGGCCCACAAT-3'

5'-GTATCTGTGGTATACAATACGTCGACCGGCAA-3'

Complementary strand:

5'-TTGCCGGTCGACGTATT-3'

5'-GTATACCACAGATACATTGTGGGCCCTTGC-3'

5'-ATACTCCACCCTTTCGCCCACAACACTCAC-3'

5'-TACACTTGGCTTGTGTTTGAGGATCAGTTG-3'

5'-TAAAGCAAGGACCACACCTTGCTGCAGGCGA-3"

The primers at the 5' and 3' ends of the coding sequence contained Pst I, and Sal I restriction sites (underlined), respectively, which were used to allow subsequent cloning of the ButaIT gene into the expression vector pGAPZαB. All primers were individually 5'-phosphorylated using enzyme T4 polynucleotide kinase. An equimolar solution of phosphorylated primers in standard T4-DNA ligase buffer (without adenosine triphosphate; ATP or dithiothreitol; DTT), was prepared in a final volume of 20 μl. The mix was boiled for 10 min, to denature secondary structures, and slowly cooled to room temperature to allow primers to anneal. After addition of ATP, DTT and DNA ligase, a ligation reaction was carried out for 24 h at 16°C. A PCR reaction was then performed, using sense and antisense primers at the 5' and 3' ends of the ButaIT gene, to obtain sufficient DNA for cloning into the intermediate vector PCR 2.1. The resulting clones were verified by sequencing. Subsequently, the ButaIT coding sequence was excised as a Pst I/Sal I fragment from PCR2.1, and cloned into the expression vector pGAPZαB to create ButaIT- pGAPZαB.

To create a construct encoding the ButaIT/GNA fusion protein, the sequence encoding the mature GNA peptide (105 residues), derived from LECGNA2 cDNA [15] (Genbank [A18023]), was excised from a previously generated construct (SFI1/GNA-pGAPZαA, [11]) by restriction digestion (5' Not I/3' Xba I) and ligated into ButaIT-pGAPZαB, which had been digested with the same enzymes. The expressed protein derived from this construct is described in fig. 1.

The construct for expressing recombinant GNA as a control also contained amino acids 1–105 of the mature GNA polypeptide (previously shown to produce a fully active lectin) inserted as an EcoR I – Xba I fragment in the vector pGAPZαA; the expressed protein, after post-translational cleavage of the vector-encoded α-factor prepro-sequence was predicted to contain two extra residues at the N-terminus (EF...) and one at the C-terminus (...D); these extra residues have been shown not to affect activity.

Expression and purification of ButaIT and ButaIT/GNA

Constructs for expressing recombinant proteins were transformed into P. pastoris (SMD1168H strain; ButaIT and ButaIT/GNA or X33 strain; GNA) according to the protocols supplied by Invitrogen [16]. Transformants were selected by plating on media containing zeocin (100 μg/ml). Selected colonies were picked off and confirmed as positive transformants by colony PCR using gene-specific primers. Clones expressing recombinant proteins were identified by immuno-dot blot or Western analysis of supernatants from small-scale cultures, using an anti-(His)₆ antibody (Invitrogen) for ButaIT, and anti-GNA antibodies for ButaIT/GNA.

For protein production, P. pastoris cells containing the ButaIT, ButaIT/GNA or GNA constructs were grown in shake flasks (30°C with shaking), or a BioFlo 110 laboratory fermenter [21] as previously described [11], except that the pH of the growing culture was maintained at 4.0. Recombinant protein samples were purified using hydrophobic interaction chromatography on a phenyl-Sepharose (Amersham-Pharmacia) column, as previously described [11]. Recombinant ButaIT, ButaIT/GNA and GNA eluted at low salt concentration, or in water, and were analysed for purity by SDS-PAGE. Lyophilised ButaIT/GNA and GNA were further purified using gel filtration on a Sephacryl S-200 column (1.6 cm diameter, 90 cm length, 0.3 ml/min), equilibrated in PBS buffer. Fractions containing purified ButaIT/GNA or GNA were pooled, analysed for purity and concentration (absorbance 280 nm; SDS-PAGE), prior to use in injection and diet bioassays. Purified proteins were de-salted by dialysis and freeze-dried, or de-salted and concentrated using Microsep TM centrifugal concentrators (VivaScience AG, Hannover, Germany).

Proteins were analysed routinely by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were prepared by adding 4× SDS sample buffer (containing 10% 2-mercaptoethanol) and boiled for 5–10 min prior to loading. For separation of low molecular weight polypeptides, SDS-PAGE was carried out using a Tris-Tricine buffer system according to the protocol of Schagger and von Jagow [22]. Gels were either stained with Coomassie Blue, or transferred to nitrocellulose using a Biorad Trans-blot SD semi-dry transfer cell, according to the manufacturers recommendations.

Concentrations of purified recombinant proteins were estimated by comparison with known amounts of standard GNA after SDS-PAGE, or Western blotting using anti-GNA antibodies (1:3300 dilution) as previously described in detail [23]. Haemagglutination assays were carried out as described previously [13].

Insect Bioassays

Partially purified ButaIT and purified ButaIT/GNA and GNA were tested for biological activity by injecting 5–10 μl of aqueous samples (freeze-dried protein re-suspended in water or PBS) into fifth stadium L. oleracea larvae of approx. 50–70 mg in weight. For each concentration tested 10–15 larvae were injected and toxic effects monitored over the next 7 days. PBS or water were injected as negative controls; no difference was observed between these two treatments, and survival over 2–3 days after the injection was routinely >90%.

A potato leaf-based artificial diet [24] was used in assays of the toxicity of recombinant ButaIT/GNA on oral delivery to lepidopteran larvae. For each treatment 20 newly moulted third stadium L. oleracea larvae were maintained in clear plastic pots containing moist filter paper to prevent diet desiccation. Survival was monitored daily. Total larval weights (± 0.1 mg) per treatment were recorded daily for the first 4 days of the assay and subsequently, individual larval wet weights (± 0.1 mg) were recorded daily, and diet consumption (per replicate) was estimated on a wet weight basis. The amount of recombinant ButaIT/GNA and GNA added to diets was estimated as described in section 2.4; control diets contained no added protein.

Toxicity of proteins when fed to rice brown planthopper was assayed using a liquid diet. An artificial diet (MMD-1), suitable for the short-term maintenance of N. lugens, was prepared according to Mitsuhashi [25]. Assays were set up by placing five second instar N. lugens nymphs in each feeding chamber, consisting of the base of a 35 mm petri dish lined with moist filter paper. Two layers of Parafilm M^® were stretched over the top, with 100 μl of artificial diet sandwiched between the two layers [26]. Feeding chambers were kept at 28°C, 90 % RH, 16 h:8 h light:dark regime, and the diet sachets were replaced every 2 days to avoid contamination. A total of 10 replicates (50 insects) were used per treatment, and survival was recorded daily over 8 days. The amounts of recombinant ButaIT/GNA and GNA added to diets was estimated as described in section 2.4; diet containing no added protein was used as negative control, and feeding chambers in which no diet was available, but insects were kept moist ("water only") were used as a positive control.

Analysis of haemolymph and gut tissue in L. oleracea larvae exposed to recombinant proteins

Haemolymph samples were extracted from L. oleracea larvae injected with recombinant proteins, or exposed to artificial diet containing ButaIT/GNA or GNA, as previously described [10]. Protein concentrations were estimated by a microtitre-based Bradford assay (Biorad) using BSA as the standard protein. Aliquots of haemolymph were analysed for the presence of GNA immunoreactivity by Western blotting as described previously. Crude gut extracts were also prepared from larvae exposed to ButaIT/GNA diets to verify binding of the fusion protein to the gut epithelium. Whole guts, dissected over ice, were flushed with PBS to remove contents. Following homogenisation, samples were centrifuged (12 000 × rpm, 4°C for 20 mins) and the supernatant assayed for protein concentration prior to analysis by Western blotting. Diet samples were also prepared (as described for gut samples) for analysis by Western blotting to confirm that fusion protein incorporated into artificial diet remained intact in 2–3 day old diet.

Statistical analyses

All data analysis was carried out using the Statview (v. 5.0; SAS Inc., Carey, NC, USA) software packages on Apple Macintosh computers. Unpaired t-tests, ANOVA analysis (Bonferroni-Dunn), Mann-Whitney non-parametric tests and survival analyses were carried out to determine any significant differences between treatments in the parameters measured.