BACKGROUND
scorpion venoms are a rich source of polypeptides with diverse biological activities, including many neurotoxins that exert their action via target-specific modulation of ion channel function .
among the well-characterised peptide toxins are those derived from the venom of scorpions belonging to the family buthidae.
these toxins are classified into two groups, based on their molecular size and activity.
the first group contains short toxins , with 3– <dig> disulphide bridges, which principally affect voltage-dependent potassium channels and conductance calcium-activated potassium channels  <cit> .
the second group contains longer toxins , cross-linked by  <dig> disulphide bridges, which mainly effect voltage-dependent sodium channels of excitable cells  <cit> .
despite the similarity of invertebrate and vertebrate ion channels, many scorpion neurotoxins exhibit specificity towards particular species or groups of species.
this specificity of action gives scorpion toxins, and other proteins with similar properties, significant potential in the development of safer alternatives to broad spectrum insecticides.
recombinant baculovirus expressing scorpion toxins have been proposed as insect control agents, and have given promising results in laboratory, glasshouse and field, although this has not led to wide-scale adoption of the technology .
the isolation and characterization of a novel short, lepidopteran-selective toxin  from the venom of the south indian red scorpion  was reported by wudayagiri et al.
<cit> .
injection of heliothis virescens with butaita  was shown to cause larval mortality through progressive, irreversible, flaccid paralysis at a dose of  <dig> μg/ <dig> mg, but was non-toxic to blowfly larvae at the same dose, or mice at a dose of  <dig> μg/g body weight.
the mature form of butait is a single polypeptide of  <dig> amino acid residues, cross-linked by four disulfide bridges, that exhibits high sequence similarity to other short toxins.
the sequence of a cdna encoding a precursor form of butait  has been lodged in the databases ; the precursor is a polypeptide of  <dig> amino acids, which is modified by removal of a 24-residue n-terminal signal peptide, and a single residue from the c-terminus, to give the mature protein.
snowdrop lectin  has been shown to bind to the gut epithelium when fed to insects, and can be detected in the circulatory system  <cit> .
the use of gna as a carrier to transport fused proteins to the haemolymph of target species after oral ingestion has been demonstrated, and can result in insecticidal effects not shown by ingestion of either the lectin or the fused protein separately.
when a fusion protein containing gna and a c-terminally fused insect neuropeptide  was fed to lepidopteran larvae, reductions in survival, and in growth and feeding, were observed  <cit>  in addition, fusion protein could be detected in the larval haemolymph by immunoassay.
similarly, gna has been used to deliver an n-terminally fused insect-specific neurotoxin, sfl <dig>  derived from the venom glands of the spider segestria florentina, to the blood of target species, causing insecticidal effects  <cit> .
this paper reports on the production, purification and biological activities of a recombinant fusion protein, butait/gna, containing the butait polypeptide fused to the n-terminus of snowdrop lectin, as a means of delivering butait to insects via oral ingestion.
the butait/gna fusion protein was toxic towards lepidopteran larvae both when injected into the haemolymph, and when fed in artifical diet.
direct evidence for the delivery of intact butait/gna to the haemolymph of orally-fed insects is presented.
in contrast to the earlier results showing that butait was specifically toxic to lepidopteran insects, the butait/gna fusion protein was found to be toxic towards insects of different orders.
RESULTS
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  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  <dig> 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  <dig> –  <dig> of mature gna via a  <dig> amino acid linker peptide .
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  <dig> amino acid c-terminal extension, containing the  <dig> tag, and is predicted to have  <dig> 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  <dig> amino acids, including a myc epitope tag and the  <dig> tag .
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  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 .
the partially purified material contained a polypeptide of estimated mol.
wt.
approx.
<dig>  as the major component ; when a gel separation was analysed by western blotting, and probing with with anti- <dig> 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– <dig> 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 .
analysis of purified butait/gna by sds-page  shows one major component of approx.
<dig>  kda, which is close to the predicted molecular weight of  <dig>  kda.
a minor component  of approx.
<dig> kda is also present.
both bands were immunoreactive towards anti-gna antibodies , but only the major component reacted with anti- <dig> 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 , 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  of proteolytic cleavage.
proteolysis occurred predominantly at or near the linker region between butait and gna, and generated a polypeptide, mol.
wt.
approx.
<dig> , which reacted with anti-gna and anti- <dig> 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  <dig>  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 .
injection of the partially purified recombinant protein at doses of the order of 1– <dig> μg caused a decline in larval suvival over a period of  <dig> days, to less than 30%.
control survival  was >70% over  <dig> days.
the effect was dose-dependent, and at high doses of butait 100% mortality was observed after  <dig> 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 5th stadium l. oleracea larvae.
the major effect on mortality was seen during the first two days after injection, after which survival  stabilised.
the effect was dose dependent; injection of  <dig> μg of fusion protein per larva  reduced survival to 40% after  <dig> days, whereas injection of  <dig> μg of fusion per larva  reduced survival to 60%.
control survival was 97% over this interval .
injection of gna at  <dig> μg per larva  had no effect on survival, which was 95% over  <dig> days ; 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 .
the effect was variable, but in a representative assay injection of approx.
<dig> μg per g insect of the fusion protein reduced weight gain over  <dig> days by approx.
40% .
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  <dig> % and  <dig> % 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  <dig> % of total protein declined significantly after day  <dig> of the assay, so that only 75% of larvae remained alive after  <dig> 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  <dig> 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  <dig> of the bioassay , so that by day  <dig> of the bioassay the mean weight of surviving butait/gna-fed larvae  was approx.
<dig> % less than that recorded for control insects .
the consumption of diet correlated with larval growth, so that overall, butait/gna at  <dig>  % of total protein caused a reduction of approx.
<dig> % of total diet consumed, compared to control treatment, and butait/gna at  <dig>  % 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 .
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  <dig>  % of dietary protein for  <dig> h and  <dig> days, and from larvae exposed butait/gna at  <dig>  % of dietary protein for  <dig> days.
typically samples were pooled from  <dig> insects to give  <dig> –  <dig> 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.
<dig> 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  <cit> , 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 .
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– <dig> days .
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 .
the fusion protein was fed to insects by incorporation into liquid diet at a concentration of  <dig> % , and its effects on survival were compared to gna at a similar concentration.
results are shown in fig.
<dig>  control survival over the 8-day bioassay was approx.
40%, and at day  <dig>  when all insects on the "water only" control had died, was approx.
50%.
corrected mortality values, based on day  <dig> of the assay, were  <dig> ± 11% for gna and  <dig> ± 5% for butait/gna; these are significantly different at p <  <dig>  .
at all times after day  <dig> 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 .
discussion
butait belongs to the sequence family of "short" scorpion toxins, which have a common pattern of cysteine residues and are thought to share a common core structure, containing one α-helix and three β-strands  <cit> .
on the basis that there is more sequence variability at the c-terminus of these proteins than at the n-terminus, where a completely conserved cysteine residue is always amino acid residue  <dig>  the butait/gna fusion protein was designed with the scorpion toxin n-terminal to the lectin, with the expectation that this orientation would be more likely to result in the fusion protein retaining toxicity.
previous fusion proteins based on gna have shown that the lectin functionality is retained in both n- and c-terminal fusions  <cit> .
expression of the toxin itself as a recombinant protein containing a c-terminal fusion "tag" sequence would confirm that c-terminal extensions to butait did not abolish toxicity.
the availability of a complete cdna sequence encoding a precursor of butait made assembling a synthetic coding sequence for the mature protein a straightforward exercise.
the high level of cysteine in butait, which is assumed to contain  <dig> cys-cys cross-links in a 37-residue protein, required that an expression system capable of correctly forming cys-cys cross-links was used to express the recombinant protein.
the use of pichia pastoris as an expression host has proved successful in our hands for expressing other cysteine-rich cross-linked proteins, such as the spider venom toxin sfli  <cit> , in a soluble, functional form, and is also able to produce functional, soluble plant lectins  <cit> .
p. pastoris was thus used as expression host for both butait and the butait/gna fusion, with both recombinant proteins engineered for secretion into culture supernatant.
comparison of the toxicity of recombinant butait with the material purified from scorpion venom by hplc  <cit>  was not possible, since only partially purified recombinant toxin was available.
however, the fusion protein was fully purified and the delivered dose could be properly quantified.
the butait purified from scorpion venom caused induction of progressive, irreversible paralysis in heliothis virescens larvae  <dig> min –  <dig> h after injection at a dose of  <dig> μg/ <dig> mg insect; in comparison, injection of butait/gna at  <dig> –  <dig> μg/ <dig> mg insect in l. oleracea larvae caused mortalities of 40–60%.
these data suggest that the toxicity of butait in the fusion protein may be compromised to some extent, but is not abolished, and the fusion protein is still an effective toxin.
previous studies using recombinant fusion proteins combining snowdrop lectin  linked either to the insect neuropeptide  or to an insect spider venom neurotoxin  have demonstrated that gna can be utilised as a transporter to deliver linked peptides to the haemolymph of lepidopteran l. oleracea larvae  <cit> .
the results presented in this paper provide further verification of the use of gna for the oral delivery of haemolymph active toxins to the blood of insects.
evidence for binding of the fusion protein to the gut and subsequent delivery to the blood was provided by western analysis of samples taken from fusion-fed larvae.
gna-immunoreactive protein with a molecular weight consistent with the recombinant butait/gna standard was seen to be present in both gut and haemolymph samples from fusion-fed l. oleracea larvae.
a degree of proteolytic degradation of the fusion was denoted by the increased intensity of an immunoreactive band of the same size as gna, concurrent with the decreased intensity of the band corresponding to butait/gna.
the degradation of ingested proteins is part of the digestive process.
whilst gna itself is resistant to gut proteolysis, proteolytic cleavage of linker regions, or polypeptides fused to gna is to be expected and has been observed previously  <cit> .
nevertheless, sufficient biologically active butait was delivered to the blood to produce detrimental effects upon larvae exposed to diet containing the fusion protein.
previous studies with sfli/gna, a fusion between a spider neurotoxin  from s. florentina and gna, also demonstrated in vivo biological activity by injection of l. oleracea larvae.
sfli/gna was seen to cause  <dig> % mortality  <dig> hours after injection of a dose of  <dig> –  <dig> μg/g insect  <cit> .
this level of toxicity is slightly higher than the effects of butait/gna on injection.
sfli/gna also showed higher toxicity than butait/gna when fed to larvae in diet, with 20% mortality over a 4-day period and an almost complete inhibition of growth when fed at levels comparable to those for butait/gna.
when sfli/gna was fed at lower levels, approx.
1% of total protein, it had no effect on survival, but caused a decrease in weight gain similar to that observed for butait/gna at approximately  <dig> % of total protein.
these results are most easily explained by a difference in sensitivity of l. oleracea larvae to the two toxins, sfli and butait, with sfli being more potent.
in both cases the analysis of haemolymph after feeding fusion protein showed that intact fusion protein was present as a result of gna-mediated transport across the gut epithelium, and thus oral toxicity would depend on the relative activity of the toxin parts of the two fusion proteins.
gna itself has no acute toxic effects in short-term feeding assays on well-developed larvae.
the bioassays in which butait/gna was fed to n. lugens show unambiguously that the fusion protein is more toxic to these insects than gna alone.
the recombinant protein used for these assays shows a similar level of toxicity to previous assays using gna purified from galanthus nivalis bulbs  <cit> .
the characterisation of butait previously reported  <cit>  suggested that this toxin is lepidopteran-specific, on the basis of non-toxicity to blowfly larvae.
the present results suggest that the specificity of butait may be broader, but could also be interpreted as an effect resulting from the fusion of the toxin to gna.
injection assays carried out with a range of insects have shown that the butait/gna fusion protein is toxic to other non-lepidopteran insects , and further work will be required to define the range of pest species for which this fusion protein may be an effective insecticide.
CONCLUSIONS
the ability of snowdrop lectin  to act as a carrier protein to direct the transport of attached molecules into the insect haemolymph has been demonstrated by the production of a recombinant protein containing the lepidopteran-specific toxin from the south indian red scorpion   fused n-terminally to snowdrop lectin.
the fusion protein  was purified to more than  <dig> % homogeneity by hydrophobic interaction chromatography and gel filtration chromatography.
biological activity was confirmed by mortality of larvae of tomato moth  injected with butait/gna, as compared to controls injected with buffer or gna  or recombinant butait .
the fusion protein showed insecticidal activity when delivered orally to l. oleracea larvae; dietary butait/gna caused a reduction in survival  and a significant reduction in larval growth and consumption, as compared to controls.
intact butait/gna was present in the haemolymph of insects fed on diet containing the fusion protein, showing that transport from the gut had occurred.
the butait/gna fusion protein was also significantly more toxic than gna alone when fed to the homopteran plant pest nilaparvata lugens , suggesting that the butait toxin may have a broader insecticidal specificity than initially thought.
fusion proteins of this type have potential applications in agriculture, both as insecticide sprays that can be applied exogenously to crops, and as gene products that can be synthesised endogenously by transgenic plants.

 scorpion venoms are a rich source of polypeptides with diverse biological activities, including many neurotoxins that exert their action via target-specific modulation of ion channel function. among the well-characterised peptide toxins are those derived from the venom of scorpions belonging to the family buthidae. these toxins are classified into two groups, based on their molecular size and activity. the first group contains short toxins, with 3– disulphide bridges, which principally affect voltage-dependent potassium channels and conductance calcium-activated potassium channels. the second group contains longer toxins, cross-linked by disulphide bridges, which mainly effect voltage-dependent sodium channels of excitable cells. despite the similarity of invertebrate and vertebrate ion channels, many scorpion neurotoxins exhibit specificity towards particular species or groups of species. this specificity of action gives scorpion toxins, and other proteins with similar properties, significant potential in the development of safer alternatives to broad spectrum insecticides. recombinant baculovirus expressing scorpion toxins have been proposed as insect control agents, and have given promising results in laboratory, glasshouse and field, although this has not led to wide-scale adoption of the technology. the isolation and characterization of a novel short, lepidopteran-selective toxin from the venom of the south indian red scorpion was reported by wudayagiri et al.. injection of heliothis virescens with butaita was shown to cause larval mortality through progressive, irreversible, flaccid paralysis at a dose of μg/ mg, but was non-toxic to blowfly larvae at the same dose, or mice at a dose of μg/g body weight. the mature form of butait is a single polypeptide of amino acid residues, cross-linked by four disulfide bridges, that exhibits high sequence similarity to other short toxins. the sequence of a cdna encoding a precursor form of butait has been lodged in the databases; the precursor is a polypeptide of amino acids, which is modified by removal of a 24-residue n-terminal signal peptide, and a single residue from the c-terminus, to give the mature protein. snowdrop lectin has been shown to bind to the gut epithelium when fed to insects, and can be detected in the circulatory system. the use of gna as a carrier to transport fused proteins to the haemolymph of target species after oral ingestion has been demonstrated, and can result in insecticidal effects not shown by ingestion of either the lectin or the fused protein separately. when a fusion protein containing gna and a c-terminally fused insect neuropeptide was fed to lepidopteran larvae, reductions in survival, and in growth and feeding, were observed in addition, fusion protein could be detected in the larval haemolymph by immunoassay. similarly, gna has been used to deliver an n-terminally fused insect-specific neurotoxin, sfl derived from the venom glands of the spider segestria florentina, to the blood of target species, causing insecticidal effects. this paper reports on the production, purification and biological activities of a recombinant fusion protein, butait/gna, containing the butait polypeptide fused to the n-terminus of snowdrop lectin, as a means of delivering butait to insects via oral ingestion. the butait/gna fusion protein was toxic towards lepidopteran larvae both when injected into the haemolymph, and when fed in artifical diet. direct evidence for the delivery of intact butait/gna to the haemolymph of orally-fed insects is presented. in contrast to the earlier results showing that butait was specifically toxic to lepidopteran insects, the butait/gna fusion protein was found to be toxic towards insects of different orders.  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 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 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 – of mature gna via a amino acid linker peptide. 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 amino acid c-terminal extension, containing the tag, and is predicted to have 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 amino acids, including a myc epitope tag and the tag. 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 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. the partially purified material contained a polypeptide of estimated mol. wt. approx. as the major component; when a gel separation was analysed by western blotting, and probing with with anti- 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– 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. analysis of purified butait/gna by sds-page shows one major component of approx. kda, which is close to the predicted molecular weight of kda. a minor component of approx. kda is also present. both bands were immunoreactive towards anti-gna antibodies, but only the major component reacted with anti- 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, 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 of proteolytic cleavage. proteolysis occurred predominantly at or near the linker region between butait and gna, and generated a polypeptide, mol. wt. approx., which reacted with anti-gna and anti- 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 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. injection of the partially purified recombinant protein at doses of the order of 1– μg caused a decline in larval suvival over a period of days, to less than 30%. control survival was >70% over days. the effect was dose-dependent, and at high doses of butait 100% mortality was observed after 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 5th stadium l. oleracea larvae. the major effect on mortality was seen during the first two days after injection, after which survival stabilised. the effect was dose dependent; injection of μg of fusion protein per larva reduced survival to 40% after days, whereas injection of μg of fusion per larva reduced survival to 60%. control survival was 97% over this interval. injection of gna at μg per larva had no effect on survival, which was 95% over days; 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. the effect was variable, but in a representative assay injection of approx. μg per g insect of the fusion protein reduced weight gain over days by approx. 40%. 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 % and % 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 % of total protein declined significantly after day of the assay, so that only 75% of larvae remained alive after 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 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 of the bioassay, so that by day of the bioassay the mean weight of surviving butait/gna-fed larvae was approx. % less than that recorded for control insects. the consumption of diet correlated with larval growth, so that overall, butait/gna at % of total protein caused a reduction of approx. % of total diet consumed, compared to control treatment, and butait/gna at % 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. 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 % of dietary protein for h and days, and from larvae exposed butait/gna at % of dietary protein for days. typically samples were pooled from insects to give – 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. 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, 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. 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– days. 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. the fusion protein was fed to insects by incorporation into liquid diet at a concentration of %, and its effects on survival were compared to gna at a similar concentration. results are shown in fig. control survival over the 8-day bioassay was approx. 40%, and at day when all insects on the "water only" control had died, was approx. 50%. corrected mortality values, based on day of the assay, were ± 11% for gna and ± 5% for butait/gna; these are significantly different at p <. at all times after day 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. discussion butait belongs to the sequence family of "short" scorpion toxins, which have a common pattern of cysteine residues and are thought to share a common core structure, containing one α-helix and three β-strands. on the basis that there is more sequence variability at the c-terminus of these proteins than at the n-terminus, where a completely conserved cysteine residue is always amino acid residue the butait/gna fusion protein was designed with the scorpion toxin n-terminal to the lectin, with the expectation that this orientation would be more likely to result in the fusion protein retaining toxicity. previous fusion proteins based on gna have shown that the lectin functionality is retained in both n- and c-terminal fusions. expression of the toxin itself as a recombinant protein containing a c-terminal fusion "tag" sequence would confirm that c-terminal extensions to butait did not abolish toxicity. the availability of a complete cdna sequence encoding a precursor of butait made assembling a synthetic coding sequence for the mature protein a straightforward exercise. the high level of cysteine in butait, which is assumed to contain cys-cys cross-links in a 37-residue protein, required that an expression system capable of correctly forming cys-cys cross-links was used to express the recombinant protein. the use of pichia pastoris as an expression host has proved successful in our hands for expressing other cysteine-rich cross-linked proteins, such as the spider venom toxin sfli, in a soluble, functional form, and is also able to produce functional, soluble plant lectins. p. pastoris was thus used as expression host for both butait and the butait/gna fusion, with both recombinant proteins engineered for secretion into culture supernatant. comparison of the toxicity of recombinant butait with the material purified from scorpion venom by hplc was not possible, since only partially purified recombinant toxin was available. however, the fusion protein was fully purified and the delivered dose could be properly quantified. the butait purified from scorpion venom caused induction of progressive, irreversible paralysis in heliothis virescens larvae min – h after injection at a dose of μg/ mg insect; in comparison, injection of butait/gna at – μg/ mg insect in l. oleracea larvae caused mortalities of 40–60%. these data suggest that the toxicity of butait in the fusion protein may be compromised to some extent, but is not abolished, and the fusion protein is still an effective toxin. previous studies using recombinant fusion proteins combining snowdrop lectin linked either to the insect neuropeptide or to an insect spider venom neurotoxin have demonstrated that gna can be utilised as a transporter to deliver linked peptides to the haemolymph of lepidopteran l. oleracea larvae. the results presented in this paper provide further verification of the use of gna for the oral delivery of haemolymph active toxins to the blood of insects. evidence for binding of the fusion protein to the gut and subsequent delivery to the blood was provided by western analysis of samples taken from fusion-fed larvae. gna-immunoreactive protein with a molecular weight consistent with the recombinant butait/gna standard was seen to be present in both gut and haemolymph samples from fusion-fed l. oleracea larvae. a degree of proteolytic degradation of the fusion was denoted by the increased intensity of an immunoreactive band of the same size as gna, concurrent with the decreased intensity of the band corresponding to butait/gna. the degradation of ingested proteins is part of the digestive process. whilst gna itself is resistant to gut proteolysis, proteolytic cleavage of linker regions, or polypeptides fused to gna is to be expected and has been observed previously. nevertheless, sufficient biologically active butait was delivered to the blood to produce detrimental effects upon larvae exposed to diet containing the fusion protein. previous studies with sfli/gna, a fusion between a spider neurotoxin from s. florentina and gna, also demonstrated in vivo biological activity by injection of l. oleracea larvae. sfli/gna was seen to cause % mortality hours after injection of a dose of – μg/g insect. this level of toxicity is slightly higher than the effects of butait/gna on injection. sfli/gna also showed higher toxicity than butait/gna when fed to larvae in diet, with 20% mortality over a 4-day period and an almost complete inhibition of growth when fed at levels comparable to those for butait/gna. when sfli/gna was fed at lower levels, approx. 1% of total protein, it had no effect on survival, but caused a decrease in weight gain similar to that observed for butait/gna at approximately % of total protein. these results are most easily explained by a difference in sensitivity of l. oleracea larvae to the two toxins, sfli and butait, with sfli being more potent. in both cases the analysis of haemolymph after feeding fusion protein showed that intact fusion protein was present as a result of gna-mediated transport across the gut epithelium, and thus oral toxicity would depend on the relative activity of the toxin parts of the two fusion proteins. gna itself has no acute toxic effects in short-term feeding assays on well-developed larvae. the bioassays in which butait/gna was fed to n. lugens show unambiguously that the fusion protein is more toxic to these insects than gna alone. the recombinant protein used for these assays shows a similar level of toxicity to previous assays using gna purified from galanthus nivalis bulbs. the characterisation of butait previously reported suggested that this toxin is lepidopteran-specific, on the basis of non-toxicity to blowfly larvae. the present results suggest that the specificity of butait may be broader, but could also be interpreted as an effect resulting from the fusion of the toxin to gna. injection assays carried out with a range of insects have shown that the butait/gna fusion protein is toxic to other non-lepidopteran insects, and further work will be required to define the range of pest species for which this fusion protein may be an effective insecticide.  the ability of snowdrop lectin to act as a carrier protein to direct the transport of attached molecules into the insect haemolymph has been demonstrated by the production of a recombinant protein containing the lepidopteran-specific toxin from the south indian red scorpion fused n-terminally to snowdrop lectin. the fusion protein was purified to more than % homogeneity by hydrophobic interaction chromatography and gel filtration chromatography. biological activity was confirmed by mortality of larvae of tomato moth injected with butait/gna, as compared to controls injected with buffer or gna or recombinant butait. the fusion protein showed insecticidal activity when delivered orally to l. oleracea larvae; dietary butait/gna caused a reduction in survival and a significant reduction in larval growth and consumption, as compared to controls. intact butait/gna was present in the haemolymph of insects fed on diet containing the fusion protein, showing that transport from the gut had occurred. the butait/gna fusion protein was also significantly more toxic than gna alone when fed to the homopteran plant pest nilaparvata lugens, suggesting that the butait toxin may have a broader insecticidal specificity than initially thought. fusion proteins of this type have potential applications in agriculture, both as insecticide sprays that can be applied exogenously to crops, and as gene products that can be synthesised endogenously by transgenic plants. 
recombinant proteins containing gna expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of gna-mediated uptake. recombinant butait and butait/gna were acutely toxic when injected into larvae of tomato moth, causing slow paralysis, leading to mortality or decreased growth. both recombinant proteins were produced using the yeast pichia pastoris as an expression host, and purified. snowdrop lectin is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. butait/gna was chronically toxic when fed to l. oleracea larvae, causing decreased survival and weight gain under conditions where gna alone was effectively non-toxic. fusion proteins based on plant lectins have potential applications in crop protection, both as exogenously applied treatments and as endogenous products in transgenic plants. butait/gna was significantly more toxic that gna alone when fed to the homopteran nilaparvata lugens in liquid artificial diet. a gene encoding a toxin, butait, from the red scorpion was synthesised and assembled into expression constructs. intact butait/gna was detected in larval haemolymph from insects fed the fusion protein orally, demonstrating transport of the linked polypeptide across the gut. the butait/gna recombinant fusion protein is toxic to lepidopteran larvae both when injected and when fed orally, showing the utility of gna as a carrier to transport potentially toxic peptides and proteins across the insect gut. proteolysis of the fusion protein was also observed. although butait has been claimed to be lepidopteran-specific, the fusion protein has more wide-ranging insecticidal activity.
 despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. snowdrop lectin is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. recombinant proteins containing gna expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of gna-mediated uptake.  a gene encoding a toxin, butait, from the red scorpion was synthesised and assembled into expression constructs. one construct contained butait alone, whereas the other contained butait fused n-terminally to a gna polypeptide. both recombinant proteins were produced using the yeast pichia pastoris as an expression host, and purified. recombinant butait and butait/gna were acutely toxic when injected into larvae of tomato moth, causing slow paralysis, leading to mortality or decreased growth. butait/gna was chronically toxic when fed to l. oleracea larvae, causing decreased survival and weight gain under conditions where gna alone was effectively non-toxic. intact butait/gna was detected in larval haemolymph from insects fed the fusion protein orally, demonstrating transport of the linked polypeptide across the gut. proteolysis of the fusion protein was also observed. butait/gna was significantly more toxic that gna alone when fed to the homopteran nilaparvata lugens in liquid artificial diet.  the butait/gna recombinant fusion protein is toxic to lepidopteran larvae both when injected and when fed orally, showing the utility of gna as a carrier to transport potentially toxic peptides and proteins across the insect gut. although butait has been claimed to be lepidopteran-specific, the fusion protein has more wide-ranging insecticidal activity. fusion proteins based on plant lectins have potential applications in crop protection, both as exogenously applied treatments and as endogenous products in transgenic plants. 