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.     

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.     

Purification and characterization of a new peptide with analgesic effect from the scorpion Buthus martensi Karch.

Anew peptide, designated as Buthus martensi Karch (BmK) AngM1, with an isoelectric point (pI) of 5.8 was purified and characterized from the venom of Buthus martensi Karch. The molecular mass was calculated as 7040.5 Da from multiple-charged ions by elelctrospray ionization mass spectroscopy (ESI/MS). The complete amino acid sequence of BmK AngM1 of 64 amino acid residues was determined by automatic sequencing of N-terminal part of the native peptide and the fragments of reduced and S-carboxymethylated (RCM)-peptide degraded by Staphylococcus aureaus V(8) protease and TPCK(N-p-Tosyl-L-phenylalanine chloromethyl ketone)-treated trypsin. Bioactivity tested using mouse-twisting model showed an evident analgesic effect with 63.0% (P < 0.001) inhibition efficiency at the dose of 0.8 mg/kg, but the LD(50) was larger than 50 mg/kg. Electrophysiological studies showed that BmK AngM1 at the concentration of 1 microm obviously inhibit voltage-dependent Na(+) current (I(Na)) and voltage-dependent delayed rectifier K(+) current (I(K)) but had no effects on transient K(+) current.     

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.     

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.     

A new insect neurotoxin AngP1 with analgesic effect from the scorpion Buthus martensii Karsch: purification and characterization.

An insect toxin named BmK AngP1 was purified from the venom of the scorpion Buthus martensii Karsch (BmK). It also shows an evident analgesic effect on mice, but is interestingly devoid of mammalian toxicity. Bioassay showed that the CPU value of AngP1 was 0.01 microg/body ( approximately 30 mg) for the excitatory insect toxicity and 43.0% inhibition efficiency for analgesia at a dose of 5 mg/kg. However, even at the dosage of 10 mg/kg no detectable toxicity on mice could be found. The isoelectric point (pI) value for AngP1 was 4.0, and its molecular mass analyzed by MALDI-TOF MS was 8141.0. The first 15 N-terminal residues of AngP1 were determined by Edman degradation and showed high similarity to that of other excitatory scorpion insect toxins. The circular dichroism spectroscopy measured on a JASCO J-720 system showed that there were 10.4% alpha-helix, 46.2% beta-strand and 14.1% turn structure in this peptide. Under two conditions single crystals of AngP1 were obtained.     

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.     

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.     

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.     

Purification, characterization of two peptides from Buthus martensi Karch.

A new peptide named Martentoxin I and an analogue Martentoxin were purified and characterized from the venom of Buthus martensi Karch. Martentoxin I consisted of 36 amino acid residues with molecular mass as 3908.0 Da determined by matrix-assisted laser desorption ionization time-of-flight-MS. The amino acid sequence was determined as GLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY by Edman degradation. Martentoxin consisted of 37 amino acid residues with a molecular mass as 4055.3 Da and it showed highly sequence identity to Martentoxin I as FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY. Estimation from circular dichroism spectra indicated Martentoxin I owned 18.0% alpha-helix, 53.0% beta-sheet structure and 3.9% turn while Martentoxin contained 13.3% alpha-helix, 64.3% beta-sheet structure and 1.1% turn. The toxicity assay showed both peptides had no toxic effects on mice up to the dose of 10 mg/kg. Electrophysiological studies showed that Martentoxin I and Martentoxin at the concentration of 1 microm significantly inhibited voltage-dependent Na+ current (INa) and voltage-dependent delayed rectifier K+ current (IK) but had no effects on transient K+ current (IA). Both interactions with Na+ and K+ channels were irreversible.     

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-.     

High-level expression,purification and properties of an Endochitinase gene without signal peptide from Lecanicillium lecanii 43H in Pichia pastoris

Molecular Biology Reports - Chitinases play the key role in hydrolysis of chitin, a huge organic carbon reservoir on earth, into monomeric sugars and their eventual conversion into valuable...     

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.     

High-level expression and purification of apoaequorin.

A fairly rapid and improved method for producing large amounts of highly pure apoaequorin, the apoprotein of aequorin which emits light on binding Ca2+, is described. The method consists of fusing the gene of the outer membrane protein A (ompA) secretion signal peptide of Escherichia coli to the apoaequorin gene and expressing the fused gene in the bacterium. The expressed protein is correctly cleaved in the process of being secreted across the cell membrane into the culture medium. The apoaequorin is subsequently purified by acid precipitation and DEAE-cellulose chromatography, yielding a product of greater than 95% purity. The availability of pure apoaequorin makes possible detailed studies of the physical-chemical properties of this Ca2(+)-binding protein and allows for the preparation of pure aequorin for use in highly specific and sensitive assays for Ca2+.     

东亚钳蝎昆虫毒素研究进展

述了我国东亚钳蝎（Buthus martensii Karsch)昆虫神经毒素的分离、,纯化、性质、基因、结构、结晶及晶体学研究的概况。     

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.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

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。     

High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli

A T7 promoter-based His6-tagging vector has been constructed that directs the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at the N terminus. The vector allows overproduction and single-step purification of His6-fusion protein by immobilized metal (Ni2+) chelate affinity chromatography. The gene encoding leucyl-tRNA synthetase (leuS) was cloned into this vector and expressed in high level. The specific activity of the synthetase in the crude extract of E. coli JM109(DE3) transformant containing the His6-tagging vector with the gene leuS was approximately 110 times that of JM109(DE3) (the host strain without the vector). The overproduced His6-fusion leucyl-tRNA synthetase can be purified to homogeneity under native conditions within 2 h by one-step affinity chromatography with an overall yield of 55%. The His6-tag at the N terminus of leucyl-tRNA synthetase did not affect its aminoacylation activity or the secondary structure.     

Expression of anti-neuroexcitation peptide III of scorpion Buthus martensii Karsch BmK ANEP III in plants

Anti-neuroexcitation peptide III of Buthus martensii Karsch (BmK ANEP III) has better anti-epileptic and anticonvulsive 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 omega 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.