Expression of anti-neuroexcitation peptide (ANEP) of scorpion Buthus martensii Karsch in Escherichia coli

According to the cDNA sequence of anti-neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti-neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by a PCR method, then was cloned into expression plasmid pET28a, fused with His tag at its 3' end. When expressed in E. coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins, while most recombinant ANEP products existed in the form of insoluble inclusion bodies. Coexpression of molecular chaperones or protein disulfide isomerase could not improve its solubility. The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and Superdex 30 chromatography. In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti-neuroexcitatory activity.     

Expression of anti-neuroexcitation peptide III of scorpion <Emphasis Type=Italic>Buthus martensii</Emphasis> Karsch BmK ANEP III in plants

Anti-neuroexcitation peptide III of Buthus martensii Karsch (BmK ANEP III) has better anti-epileptic and anticonvulsant effects in the test animal models. The present study is aimed at developing transgenic tomato and tobacco lines overproducing the ANEP III protein. Using the molecular cloning technique, the plant expression vector pBI-ANEP III was constructed successfully. The ANEP III expression cassette included a double CaMV 35S promoter with Ω enhancers, the ANEP III gene with the Kozak sequence, the ER retention signal and the NOS terminator. Recombinant plasmids were transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw transformation methods. By the Agrobacterium-mediated leaf disc transformation method, tobacco (Nicotiana tabacum) and tomato (Lycopersicum esculentum) lines were transformed. Transformants were screened and confirmed by PCR, RT-PCR and western blotting analysis. It was demonstrated that the ANEP III gene was successfully expressed in the genomic DNA of transgenic plants. The ANEP III protein was detected by immunofluorescence analysis, and the results confirmed the high amount of ANEP III protein, being 0.81 and 1.08% of total soluble proteins in transgenic tobacco and tomato. The study of plants with high expression levels of ANEP III has an important theoretical and practical significance and provides valuable information for establishing a new, economical and effective system for industrial protein production.     

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.     

Functional study of active residues scorpion insect toxin BmK IT from Buthus martensii Karsch

Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating. An excitatory insect toxin, BmK IT, is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is a toxin with a non-glutamate residue at the relevant position in the excitatory scorpion β-toxin subfamily. In this study, the mutants of recombinant BmK IT (BmK IT (I25E), BmK IT (E15G), BmK IT C-terminal (TKSYCDVQIN) truncated) were achieved by site-directed mutagenesis. Biological activity of BmK IT and its mutants confirmed these residues or peptides played key roles in BmK IT. BmK IT (I25E) could increase the sensitivity of BmK IT, but BmK IT(E15G) could decrease the sensitivity of BmK IT on Sf9 cells. BmK IT truncated C-terminal hydrophobic amino acids could cross the species boundaries and was effective on mammalian C6 cells. To date, several excitatory insect toxins have been isolated and identified from the venom of Buthus martensii Karsch. However, no functional data are available and therefore its classification in the family of excitatory insect toxins remains putative and is just based on its high similarity with the other toxins of this family. These results verified I25, E15 and C-terminal (TKSYCDVQIN) in BmK IT played key roles in the interaction of the BmK IT and its receptor- sodium channels on the surface of insect cells and laid a foundation for further structural and functional analysis of BmK IT.     

Expression and purification of the BmK Mm2 neurotoxin from the scorpion Buthus martensii Karsch and its biological activity test

The gene encoding neurotoxin (BmK Mm2) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21 (DE3) at a level of 1.6 mg/L using expression plasmid pExSecI system. SDS-PAGE analysis of the cell lysate confirmed that gene BmK Mm2 was expressed in soluble form and the expressed production was secreted into Luria-Bertani (LB) culture medium from Escherichia coli. According to the characters of pExSecI expression system, the IgG binding domain-ZZ of Protein A is fused to the N-terminal of BmK Mm2. Recombinant BmK Mm2 (ZZ-BmK Mm2, pI 6.81, 22.007 kDa) was purified rapidly and efficiently by IgG-Sepharose 6 Fast Flow and Superdex-75 gel filtration chromatography, produced a single band on SDS-PAGE. Western blot analysis demonstrated that this protein was recombinant BmK Mm2. The results of MTT assay, morphological observation of nucleus and single cell gel electrophoresis showed that the expressed recombinant BmK Mm2 was toxic for glial cells of mice, which indicate that it has biological activity.     

Purification, characterization and cDNA cloning of an analgesic peptide from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU2)

In this study an analgesic peptide was purified through five continuous chromatographic steps. The mouse twisting model test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU2 was further qualified by Reverse Phase-High Performance Liquid Chromatography and High Performance Capillary Electrophoresis. The molecular weight, isoelectric point, and N-terminal sequence of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was cloned by rapid amplification of the cDNA ends from the cDNA pool of scorpion glands. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 (GenBank Acc. No. AF288608) and BmK alphaTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.     

Pharmacological comparison of two different insect models using the scorpion alpha-like toxin BmK M1 from Buthus martensii Karsch

In this study, the effect of the scorpion alpha-like toxin BmK M1 was investigated on isolated DUM neurons from Locusta migratoria and compared with the effect on para/tipE voltage-gated Na(+) channels (VGSC), cloned from Drosophila melanogaster. The two insects display different pharmacological properties regarding alpha-like toxins. Moreover, with the aid of the alpha-like toxin BmK M1 and 5 of its mutants, the importance of aromatic residues for the interaction of the toxin with the VGSC in L. migratoria and D. melanogaster, is shown.     

Expression, renaturation and functional analysis of an excitatory insect-specific toxin from scorpion Buthus martensii Karsch

The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on insect sodium channel.     

Purification and N-terminal partial sequence of anti-epilepsy peptide from venom of the scorpion Buthus martensii Karsch.

An anti-epilepsy peptide (AEP) was isolated and purified from venom of the scorpion Buthus martensii Karsch. The purification procedure included CM-Sephadex C-50 chromatography, gel filtration on Sephadex G-50 and DEAE-Sephadex A-50 chromatography. Its homogeneity was demonstrated by pH 4.3 polyacrylamide-disc-gel electrophoresis, focusing electrophoresis and SDS/polyacrylamide-disc-gel electrophoresis. The Mr of this peptide, calculated from measurements in SDS/15%-polyacrylamide-disc-gel and SDS/20%-polyacrylamide-disc-gel electrophoresis, is 8300. The isoelectric point is 8.52 by pH 8-9.5-range isoelectric focusing. No haemorrhagic or toxic activities were found. No toxicity was found even after the dose reached 28 mg/kg. The pharmacological tests showed that the AEP had no effect on heart rate, blood pressure or electrocardiogram, but strongly inhibited epilepsy induced by coriaria lactone and cephaloridine. The fluorescence spectrum showed that the peptide has a strong emission peak at 337 nm. Amino acid analysis suggested that the AEP is composed of 66 residues from 18 amino acids and has an Mr of 8290. The sequence of the first 50 N-terminal residues is as follows: Asp-Gly-Tyr-Ile-Arg-Gly-Ser-Asp-Asn-Cys-Lys-Val-Ser-Cys-Leu-Leu-Gly-Asn- Glu-Gly - Cys-Asn-Lys-Glu-Cys-Arg-Ala-Tyr-Gly-Ala-Ser-Tyr-Gly-Tyr-Cys-Trp-Thr-Val- Lys-Leu - Ala-Gln-Asp-Cys-Glu-Gly-Leu-Pro-Asp-Thr-.     

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.     

Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

BmK AngM1 is an analgesic peptide from the venom of Buthus martensii Karsch (BmK). The synthetic gene encoding BmK AngM1 was optimized on the basis of its cDNA sequence and the codon usage preference of Pichia pastoris. The codon-optimized gene was cloned into pPIC9K and then transformed into P. pastoris. SDS-PAGE and Western blot analysis showed that the recombinant BmK AngM1 (rBmK AngM1) was expressed by the addition of methanol to the medium, and its maximum production reached above 500 mg/l. The purified rBmK AngM1 could be obtained efficiently by Nickel affinity chromatography. Analgesic bioassay, by the mouse-twisting model, showed that rBmK AngM1 had evident analgesic effect with an ED₅₀ of 0.5 mg/kg.     

A depressant insect toxin with a novel analgesic effect from scorpion Buthus martensii Karsch

A new peptide named BmK dITAP3 from scorpion Buthus martensii Karsch (BmK) has been identified to possess a dual bioactivity, a depressant neurotoxicity on insects and an analgesic effect on mice. The bioassays also showed that the peptide was definitely devoid of the neurotoxicity on mammals, which indicated that the analgesic effect of BmK dITAP3 could not be ascribed to the syndromic effects of a mammalian neurotoxicity. BmK dITAP3 exhibited 43.0% inhibition efficiency of the analgesic effect on mice at a dose of 5 mg/kg and the FPU value of 0.5 microg/body (approximately 30 mg) on the fly larvae. The pI value and the molecular mass determined by MALDI-TOF MS for dITAP3 were 6.5 and 6722.7, respectively. Its first 15 N-terminal residues were determined by Edman degradation, based on which the full amino acid sequence was deduced from the cDNA sequence encoding the peptide with 3'-RACE. Circular dichroism and sequence based prediction analyses showed dITAP3 may have a similar molecular scaffold as the most scorpion toxins but with features of the more beta structures and much less of alpha helix. The details of the purification, characterization and sequencing as well as the sequence comparison with other depressant insect toxins and the correlation between the analgesic effect and the insect toxicity will be reported and discussed, respectively.     

蝎毒中枢镇痛机制的初步探讨

目的：研究BmK蝎毒是能过何种受体实现其中枢镇痛作用的;外侧隔核是否是BmK蝎毒产生中枢镇痛作用的重要部位之一。方法：用辐射热一甩尾法测定痛阈,用玻璃微电极记录束旁核的单位放电;通过不锈钢套管向侧脑室和外侧隔核内微量注射0.01％BmK蝎毒。结果：向大鼠侧脑室2μl0.01%蝎毒可明显升高痛阂。该效应可被侧脑室注射2μl0.25%纳洛酮完全翻转,向隔核内注射0.5μl0.01%BmK蝎毒,分别对束帝核中的71％（15／21）痛兴奋单位和83％（5／6）痛抑制单位对痛刺激的反应减北,而对痛无关单位的电活动夫明显影响。结论：BmK蝎毒的中枢镇痛作用可能主要是通过吗啡受体实现的;隔核是BmK蝎毒产生中枢镇痛作用的重要部位之一。     

Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series.It is medially toxic and belongs to group III α-toxins.The purified sample was crystallized in rhombic space group P61.Using an X-ray diffraction technique,the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution.The model was refined.The final crystallographic R factor was 0.142 and the free R factor was 0.173.The root mean square deviation is 0.001 5 nm for the bond length and 1.753°for the bond angles.64 water molecules were added to the asymmetric unit.The refined structure showed an unusual non-prolyl cis peptide bond at residue 10.The structure was compared with group II α-toxin BmK M8 (an acidic,weak toxin).The potential structural implications of the cis peptide bond were discussed.     

Expression of an Insect Excitatory Toxin, BmK IT, from the Scorpion, Buthus martensii Karsch, and its Biological Activity

An insect excitatory toxin from Buthus martensii Karsch (BmK IT) was cloned into the expression vector, pTWIN1, and expressed into Escherichia coli BL21 (DE3) host cells. The soluble fusion expression of CBD-intein-BmK IT was obtained. The recombinant BmK IT was purified by two anion-exchange chromatography columns and one gel chromatography column. Bioassays were carried out to verify the toxicity of this recombinant toxin. At the end of a 96 h experimental period, 83% of cotton bollworm larvae were killed with an LT(50) value of 58-62 h. Furthermore, the average weight of larvae fed on BmK IT-containing media was approx 4% of that of the control groups. The results indicate that the expressed and purified recombinant BmK IT has biological activity.     

Expression of an antitumor-analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli

The gene encoding a putative mature antitumor-analgesic peptide (AGAP) from the venom of the Chinese scorpion Buthus martensii Karsch was obtained by polymerase chain reaction (PCR) according to its cDNA sequence and expressed in Escherichia coli. While most of the recombinant AGAP was expressed in the form of insoluble inclusion body. The recombinant AGAP was purified to homogeneity by metal chelating affinity chromatography. Pharmaceutical tests showed that the recombinant AGAP has both analgesic and antitumor activities on mice.     

Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch.

For a long time Asian scorpion Buthus martensi Karsch (BmK) has been used in Chinese traditional medicine to cure many diseases of nervous system. Here we report the purification and characterization of a pharmacologically active neurotoxin from the scorpion BmK. This toxin had little toxicity in mice and insects but was found to have an anti-epilepsy effect in rats, and is thus named as BmK anti-epilepsy peptide (BmK AEP). Its amino-acid sequence was determined by lysylendopeptidase digestion, Edman degradation and mass spectrographic analysis. Based on the determined sequence, the gene coding for this peptide was also cloned and sequenced by the 3' and 5' RACE methods. It encodes a precursor of 85 amino-acid residues including a signal peptide of 21 residues, a mature peptide of 61 residues and three additional residues Gly-Lys-Lys at the C-terminus. The additional Gly sometimes followed by one or two basic residues is prerequisite for the amidation of its C-terminus. C-terminal amidation was also verified by the molecular-mass determination of BmK AEP. This anti-epilepsy peptide toxin shares homology with other depressant insect toxins. The remarkable difference between them was mainly focused at residues 6, 7 and 39; these residues might relate to the unique action of BmK AEP.     

Isolation,purification, and N-terminal partial sequence of an antitumor peptide from the venom of the Chinese scorpion Buthus martensii Karsch

An antitumor peptide (ANTP) was isolated and purified from the venom of the Chinese scorpion Buthus martensii Karsch. The purification procedure included gel filtration on Sephadex G-50 and Superdex 30 high resolution chromatography, Phenyl Sepharose 6 Fast Flow chromatography, and SP-Sepharose Fast Flow chromatography. Its homogeneity was demonstrated by size exclusion HPLC on TSK G2000 SW. The isoelectric point is more than 10 by pH 3-10 range isoelectric focusing. ANTP has a relative molecular mass of 6280, calculated from the measurement of 16.5% SDS-PAGE. The pharmacological tests showed that ANTP has antitumoral effects in the mouse S-180 fibrosarcoma model and Ehrlich ascites tumor model. Amino acid analysis suggested the ANTP is rich in glycine and does not have histidine and threonine. The sequence of the first 25 N-terminal residues is as follows: Val-Arg-Asp-Gly-Tyr-Ile-Ala-Asp-Asp-Lys-Asn-Cys-Ala-Tyr-Phe-Cys-Gly-Arg-Asn-Ala-Tyr-Cys-Asp-Asp-Glu.     

Identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from Buthus martensii Karsch

The scorpion venom peptides with no disulfide bridge are rarely identified and poorly characterized so far. Here, we report the identification and characterization of four novel disulfide-bridge-free venom peptides (BmKa1, BmKa2, BmKb1 and BmKn2) from Buthus martensii Kasch. BmKa1 and BmKa2 are very acidic and hydrophilic, showing no any similarity to other proteins, whereas BmKb1 and BmKn2 both are basic, alpha-helical peptide with an amidated C-terminus, showing a little homology with other peptides. Functional tests with synthetic peptide showed that BmKn2 has strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, whereas BmKb1 has weak activity in inhibiting the growth of these bacteria.     

Eukaryotic expression and purification of anti-epilepsy peptide of Buthus martensii Karsch and its protein interactions

To investigate the structure–function relationships of intestinal fatty acid-binding protein (I-FABP) in cellular fatty acid (FA) trafficking, we compared the distribution of a fluorescent FA analog (BODIPY FL C16) in Cos-1 cells transiently transfected with the wild type protein (wt I-FABP) to that of a variant deleted of the alpha helical domain (HL I-FABP). In vector-only cells, BODIPY fluorescence was distributed throughout the cytoplasm. In the absence of added FA, wt I-FABP was found largely in the perinuclear region with some cytoplasmic staining as well. Addition of BODIPY FL C16 to transfected cells showed that the fluorescent FA was essentially completely colocalized with the protein in the cytoplasmic and perinuclear regions as well as in cytoplasmic clusters that are not observed in the absence of wt I-FABP. For HL I-FABP, the distribution of the protein in the absence of FA was diffusely cytoplasmic, in marked contrast to the wt protein. Addition of BODIPY led to less extensive colocalization than that observed for wt I-FABP. In particular, no localization to the perinuclear region was found. Organelle colocalization studies showed that both proteins colocalized with mitochondria and endoplasmic reticulum/golgi markers, but little with a lysosomal marker. The perinuclear localization for wt I-FABP and BODIPY did not show colocalization with any of the markers tested. Taken together, these results indicate that I-FABP binds FA in vivo and that the helical domain may be important for targeting I-FABP to a perinuclear domain but not, perhaps, to the endoplasmic reticulum, golgi apparatus or mitochondria.