Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.     

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.     

植物乳杆菌环二腺苷酸合成酶的克隆表达与活性分析

【背景】环二腺苷酸(Cyclic Diadenosine Monophosphate,c-di-AMP)是一种主要存在于革兰氏阳性菌中的重要的第二信使分子,其参与细菌的生长、生存、抗逆性等多种生理活动,但目前关于乳酸菌中c-di-AMP的研究甚少。【目的】从植物乳杆菌(Lactobacillus plantarum)中克隆得到c-di-AMP合成酶基因,在大肠杆菌中进行可溶性表达并研究其体外活性。【方法】使用高效液相色谱以及质谱分析对植物乳杆菌-YRA7细胞内容物中的c-di-AMP进行检测;以植物乳杆菌-YRA7基因组DNA为模板,克隆c-di-AMP合成酶基因(lpDacA),构建重组表达载体pET-28a-lpDacA并在大肠杆菌BL21(DE3)中诱导表达,通过Ni-NTA亲和层析纯化后进行体外活性研究。【结果】在植物乳杆菌中检测到c-di-AMP分子;成功构建了c-di-AMP合成酶基因的重组表达质粒,该重组蛋白在大肠杆菌中得到可溶性表达;体外活性分析显示,该重组蛋白可以催化ATP生成c-di-AMP,其活性依赖于二价阳离子的存在,在Mg~(2+)存在以及碱性环境下活性较强;RHR是合成酶活性的关键基序,是环二腺苷酸合成酶与ATP的结合位点。【结论】植物乳杆菌c-di-AMP合成酶的克隆表达及活性分析为进一步研究c-di-AMP在植物乳杆菌中的作用奠定了基础。     

Expression,purification, and characterization of equine lactoferrin in Pichia pastoris

Lactoferrin is an 80kDa iron-binding glycoprotein. It is secreted by exocrine glands. Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it. In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector. The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column. The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin. Its N-terminal sequence was identical to that of the native ELF. The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly. The recombinant protein appears to be hyperglycosylated by the host strain, GS115. This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P. pastoris system. An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

Molecular cloning,characterization and expression of a gene encoding phosphoketolase from Termitomyces clypeatus

A phosphoketolase (pk) gene from the fungus Termitomyces clypeatus (TC) was cloned and partially characterized. Oligonucleotide primers specific for the phosphoketolase gene (pk) were designed from the regions of homologies found in the primary structure of the enzyme from other fungal sources related to TC, using multiple sequence alignment technique. The cDNA of partial lengths were amplified, cloned and sequenced in three parts by 3′ and 5′ RACE and RT-PCR using these oligonucleotide primers. The full length ds cDNA was constructed next by joining these three partial cDNA sequences having appropriate overlapping regions using Overlap Extension PCR technique. The constructed full length cDNA exhibited an open reading frame of 2487 bases and 5′ and 3′ UTRs. The deduced amino acid sequence, which is of 828 amino acids, when analyzed with NCBI BLAST, showed high similarities with the phosphoketolase enzyme (Pk) superfamily with expected domains. The part of the TC genomic DNA comprising of the pk gene was also amplified, cloned and sequenced and was found to contain two introns of 68 and 74 bases that interrupt the pk reading frame. The coding region of pk cDNA was subcloned in pKM260 expression vector in correct frame and the protein was expressed in Escherichia coli BL21 (DE3) transformed with this recombinant expression plasmid. The recombinant protein purified by His-tag affinity chromatography indicated the presence of a protein of the expected size. In vivo expression studies of the gene in presence of different carbon sources indicated synthesis of Pk specific mRNA, as expected. Phylogenetic studies revealed a common ancestry of the fungal and bacterial Pk. The TC is known to secrete several industrially important enzymes involved in carbohydrate metabolism. However, the presence of Pk, a key enzyme in pentose metabolism, has not been demonstrated conclusively in this organism. Cloning, sequencing and expression study of this gene establishes the functioning of this gene in T. clypeatus. The Pk from TC is a new source for commercial exploitation.     

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.     

Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis

The gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies. The product of the gene has been identified as the superoxide dismutase (SOD) of M. tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form. The derived amino acid sequence of M. tuberculosis SOD reveals a close similarity to manganese-containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand. SOD is present in the extracellular fluid of logarithmic-phase cultures of M. tuberculosis, but the structural gene is not preceded by a signal peptide sequence. Insertion of the M. tuberculosis SOD gene into a novel shuttle vector demonstrated the mycobacteria but is ineffective in Escherichia coli.     

Cloning, Expression, and Biological Function of a dTDP-Deoxyglucose Epimerase (gerF) Gene from Streptomyces sp. GERI-155

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules which has antimicrobial activities against gram-positive bacteria. The deoxyhexose biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-155 genome has now been isolated. Four orf were identified and a putative orf, supposed to code for the dTDP-deoxyglucose epimerase gene, was designated as gerF. gerF was expressed in E. coli using recombinant expression vector pHJ3. The recombinant protein expressed in a soluble form. The enzyme was purified by Ni-affinity column using imidazole buffer as eluents. The molecular mass of the expressed protein correlated with the predicted mass (36,000 Da) deduced from the cloned gene sequence data. The purified enzyme produced maltol from dTDP-4-keto-6-deoxyglucose and it was confirmed that the expressed protein was dTDP-deoxyglucose epimerase catalyzing epimerization of C-3 and C-5 or C-3 of dTDP-4-keto-6-deoxyglucose.     

Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae

The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.     

Cloning and expression of malarial pyrimidine enzymes

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.     

Cloning, expression, and purification of the 27 kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis

A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%.     

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫(Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定,并构建了融合表达质粒pET-ESTA2。转化大肠杆菌BL21后,在异丙基硫代牛乳糖苷(IPTG)的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达。表达产物经亲和层析纯化获得了1条带的重组蛋白。与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高。表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高。羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4- glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion- exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.      

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫（Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定。并构建了融合表达质粒pET-ESTA2,转化大肠杆菌BL21后,在异丙基硫代半乳糖苷（IPTG）的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达,表达产物经亲和层析纯化获得了1条带的重组蛋白,与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高,表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高,羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

Isocitrate dehydrogenase from Thermus aquaticus YT1: purification of the enzyme and cloning, sequencing, and expression of the gene.

Isocitrate dehydrogenase from an extremely thermophilic bacterium, Thermus aquaticus YT1, was purified to homogeneity, and the gene was cloned by using a degenerate oligonucleotide probe based on the N-terminal sequence. The gene consisted of a single open reading frame of 1,278 bp preceded by a Shine-Dalgarno ribosome binding site, and a terminator-like sequence was detected downstream of the open reading frame. The G+C content of the coding region was 65%, and that of the third nucleotide of the codons was 93%. The amino acid sequence of the enzyme showed a relatively low level of similarity to the counterpart from T. thermophilus (35% identity) but showed higher levels of similarity (54 to 69% identity) to the other bacterial counterparts so far reported, including those from Escherichia coli, Bacillus subtilis, Vibrio sp., and Anabaena sp. The cloned gene was highly expressed in E. coli and easily purified to homogeneity by heat treatment (70 degrees C, 30 min) and DEAE column chromatography to yield approximately 10 mg of protein from 1 g of wet cells. The recombinant enzyme showed high thermostability and almost the same heat denaturation profile as the intact enzyme purified from the thermophile cells, implying that the recombinant protein has the same structure as the intact one.     

人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶联表达

人降钙素 (hCT)是 32氨基酸的多肽激素 ,C-端为α脯氨酰胺结构 ,具有调节体内钙、磷代谢等许多重要生理功能。用重组昆虫杆状病毒表达系统 ,偶联表达合成的人修饰型降钙素 (hmCT)基因与GST融合基因和大鼠酰胺化酶 (PAM)基因 ,再用抗hmCT或抗PAM抗体 ,既检测到由昆虫细胞表达的GSThmCT产物也检测到PAM产物。经GSH 琼脂糖凝胶亲和层析 ,分离纯化GSThmCT融合蛋白。这种蛋白修饰酶与底物在真核细胞偶联表达也将适用于其他生物活性肽的体外表达     

Characterization of recombinant fungal phytase (phyA) expressed in tobacco leaves.

The phyA gene from Aspergillus ficuum coding for a 441-amino-acid full-length phytase was expressed in Nicotiana tabacum (tobacco) leaves. The expressed phytase was purified to homogeneity using ion-exchange column chromatography. The purified phytase was characterized biochemically and its kinetic parameters were determined. When the recombinant phytase was compared with its counterpart from Aspergillus ficuum for physical and enzymatic properties, it was found that catalytically the recombinant protein was indistinguishable from the native phytase. Except for a decrease in molecular mass, the overexpressed recombinant phytase was virtually the same as the native fungal phytase. While the temperature optima of the recombinant protein remain unchanged, the pH optima shifted from pH 5 to 4. The results are encouraging enough to open the possibility of overexpressing phyA gene from Aspergillus ficuum in other crop plants as an alternative means of commercial production of this important enzyme.