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.     

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.     

Site-Directed Mutagenesis of BmK AGP-SYPU1: The Role of Two Conserved Tyr (Tyr5 and Tyr42) in Analgesic Activity

In this study, the role of two conversed tyrosines (Tyr5 and Tyr42) from the scorpion toxin BmK AGP-SYPU1 was investigated with an effective Escherichia coli expression system. Site-directed mutagenesis was used to individually substitute Tyr5 and Tyr42 with hydrophobic or hydrophilic amino acids, and the extent to which these scorpion toxin BmK AGP-SYPU1 tyrosines contribute to analgesic activity was evaluated. The results of the mouse-twisting test showed that Tyr5 and Tyr42 are associated with the analgesic activity of the toxin because the analgesic activities of Y5F and Y42F were significantly increased compared with the rBmK AGP-SYPU1; however, the Y5W had decreased activity. The results of molecular simulation reveal the following: (1) for analgesic activity, the core domain of the scorpion toxin BmK AGP-SYPU1 is key and (2) for pharmacological function, Tyr42 is most likely involved when the core domain conformation is altered. These studies identify a new relationship between the structure and analgesic activity of the scorpion toxin BmK AGP-SYPU1 and are significant for further research and the application of analgesic peptides.     

Purification,characterization and cDNA cloning of an analgesic peptide from the chinese scorpion <Emphasis Type="Italic">Buthus martensii</Emphasis> 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 α2 (GenBank Acc. No. AF288608) and BmK αTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.     

Synthesis,Expression and Purification of a Type of Chlorotoxin-like Peptide from the Scorpion, <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch,and its Acute Toxicity Analysis

A gene, rBmK Cta, encoding a chlorotoxin-like peptide from the scorpion, Buthus martensii Karsch, was synthesized according to the sequence optimized for codon usage in Escherichia coli and was expressed in E.␣coli BL21 (DE3) using a pExSecI expression system in which the IgG-binding domain-ZZ of protein A is fused to the N-terminal of rBmK CTa. The fusion protein, ZZ-rBmK CTa, was expressed in soluble form (7.8 mg l⁻¹) and was purified to give a single band on SDS-PAGE. The domain-ZZ of fusion protein ZZ-rBmK CTa was removed by cleavage of an Asn–Gly peptide bond with hydroxylamine. The rBmK CTa was separated from the IgG-binding moiety by a second passage through the IgG affinity column. Western blot analysis demonstrated that this protein was rBmK CTa. Acute toxicity assay in mice demonstrated that the rBmK CTa had an LD₅₀ value of 4.3 mg kg⁻¹.     

Recombinant scorpion insect excitatory toxin BmK IT accelerates the growth of insect Spodoptera frugiperda 9 cells

Several scorpion insect toxins are selectively active on the lepidopterous and dipterous insects. The gene encoding insect excitatory neurotoxin (BmK IT) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21(DE3) at a high level of 3 mg/0.5 L using the prokaryotic expression system pTWIN1. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), whole-cell patch-clamp technique and immunofluorescence assays were used to evaluate the toxicity of rBmK IT to insect Spodoptera frugiperda 9 (Sf9) cells and to analyze the potential mechanism of this toxicity. rBmK IT accelerated the growth of Sf9 cells in a dose-dependent manner. Voltage-gating sodium channel activity could not be detected in Sf9 cells using a whole-cell patch-clamp technique. However, immunofluorescence analysis clearly showed co-localization of tetrodotoxin (TTX) and rBmK IT on the Sf9 cell membrane, which demonstrated that rBmK IT could bind to and act on the voltage-gated sodium channels on the Sf9 cells by the high affinity action power. The findings presented in this study are essential for further study of this peptide.     

Cloning,expression, and pharmacological activity of BmK AS,an active peptide from scorpion <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch

BmK AS is a β long-chain scorpion peptide from the venom of Buthus martensii Karsch (BmK). It was efficiently expressed as a soluble and functional peptide in Escherichia coli, and purified by metal chelating chromatography. About 4.2 mg/l purified recombinant BmK AS could be obtained. The recombinant BmK AS maintained a similar analgesic activity to the natural one in both the mouse-twisting test and hot-plate procedure. It also exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. BmK AS is the first long-chain scorpion peptide reported to have antimicrobial activity, and is a valuable molecular scaffold for pharmacological research.     

Cloning and characterization of BmK86, a novel K+ -channel blocker from scorpion venom

Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K+ -channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purified by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of alpha-KTxs. The systematic number assigned to BmK86 is alpha-KTx26.1.     

Intein介导的重组昆虫毒素BmK IT在大肠杆菌中的可溶性表达、纯化及活性分析

为获得重组蝎昆虫毒素BmKIT,通过PCR方法在BmKIT基因的3′端融合了编码6个组氨酸残基的核苷酸序列,将其插入原核表达载体pTWIN1的内含肽Ssp DnaB Intein基因下游的多克隆位点(MCS)。将获得的表达质粒转化大肠杆菌BL21(DE3)中,用IPTG诱导融合蛋白表达。用Ni-NTA亲和层析柱从菌体裂解液中纯化了CBD-Intein-BmK IThis6融合蛋白,并在柱上诱导Intein自剪切,成功去除融合子CBD-Intein。通过Superdex75凝胶过滤层析获得了纯度达95%以上的BmK IThis6蛋白,该蛋白不仅具有正确的二级结构而且有生物活性。     

Soluble expression, purification and the role of C-terminal glycine residues in scorpion toxin BmK AGP-SYPU2

The existence of glycine residues in long-chain scorpion toxins has been well documented. However, their role as analgesics has not been evaluated. To address this issue, we investigated the functional role of glycines in the C-terminal end of Chinese-scorpion toxin from Buthus martensii Karsch (BmK AGP-SYPU2) using site-directed mutagenesis and analgesic activity assays. Recombinant BmK AGP-SYPU2 and its mutants were efficiently expressed in E. coli and purified to homogeneity using immobilized metal ion affinity chromatography (IMAC) and cation exchange chromatography. The mouse-twisting test was used to detect the analgesic activity of BmK AGP-SYPU2 and its mutants. As a result, we identified glycines at the C-terminal end that, when altered, significantly affected analgesic activity. Also, Mut6566 was significantly decreased compared to BmK AGP-SYPU2. These data indicate that the glycines at the C-terminal end are important for the analgesic activity of BmK AGP-SYPU2.     

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.     

Expression and purification of an antitumor-analgesic peptide from the venom of Mesobuthus martensii Karsch by small ubiquitin-related modifier fusion in Escherichia coli

To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the antitumor-analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO-AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni-NTA affinity chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N-terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences.     

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.     

新型神经保护肽[Gly14]-Humanin的表达，纯化和活性鉴定

Humanin（HN）是近年来在人体内发现的内源性多肽,能够保护神经细胞免于各种阿尔采默病病相关因素诱导的凋亡,HN肽链第14位氨基酸被Gly取代的突变体[Gly14]-Humanin（HNG）,神经保护活性比HN高近1 000倍。但由于HNG天然获取的途径有限,限制了对其功能的进一步研究。本课题组构建了HNG的表达质粒pET32a/HNG,并将其转化大肠杆菌BL21 trxB (DE3),诱导表达后HNG以可溶性融合蛋白的形式出现并用镍离子亲和层析纯化,纯化的融合蛋白用肠激酶切割后经反相层析得到纯化的HNG多肽（23 mg每1 L细菌培养液）,重组HNG多肽的分子量经电喷雾电离质谱（ESI-MS）检测为2876.5道尔顿,其N末端8个氨基酸残基的序列经Edman降解法测定为A-M-M-A-P-R-G-F,均与理论值一致。神经细胞保护实验表明,重组HNG多肽的神经保护作用与天然的HNG相近     

Expression,purification and functional characterization of a recombinant scorpion venom peptide BmTXKbeta

BmTXKbeta, a scorpion toxin isolated from the Chinese scorpion Buthus martensii Karsch (BmK), was expressed as a GST fusion protein in BL21 (DE3) strain. The recombinant GST-BmTXKbeta protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKbeta fusion protein released an approximate 6.5kDa protein which was the expected size for correctly processed. About 2mg purified recombinant BmTXKbeta protein (rBmTXKbeta) was produced from 1l bacterial culture, using this expression and purification system. The function of rBmTXKbeta was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKbeta inhibited the transient outward current (I(to)) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKbeta prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.     

Polyclonal Antibody Against a Recombinant Chlorotoxin-like Peptide from the Chinese Scorpion and Detection of its Putative Receptors in Human Glioma Cells

The nucleotide sequence of a type of chlorotoxin-like peptide, an inhibitor of small-conductance Cl⁻ channels, from the scorpion, Buthus martensii Karsch, was synthesized (named rBmK CTa) according to the sequence optimized for codon usage in E. coli. It was over-expressed using a pExSecI expression system and purified to homogeneity. Polycolonal antibodies to the purified protein were raised in rats. Overlay assay and pull-down assay showed that this toxin specially binds to two proteins in the glioma cells with corresponding molecular weights of about 80 and 35 kDa. They may serve as candidate receptors or alternative cellular component for interaction with rBmK CTa.     

水通道蛋白1-C末端肽段/GST融合蛋白的原核表达(简报)

水通道蛋白(Aquaporin,AQP)是一类选择性高效转运水分子的细胞膜通道蛋白,广泛存在于原核和真核生物细胞的细胞膜上,主要介导自由水分子的被动跨膜转运,对保持细胞内外液环境的稳态平衡起着重要的作用.     

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.