Molecular cloning,characterization, and expression of a cDNA encoding an endochitinase gene from the mycoparasite Stachybotrys elegans

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogenic fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall degrading enzymes such as chitinases. This report details the cloning by RACE-PCR and characterization of a full-length cDNA clone, sechi44, that appears to encode an extracellular endochitinase. An analysis of the sechi44 sequence indicates that this gene contains a 1269-bp ORF and encodes a 423-aa polypeptide. The SECHI44 protein has a calculated molecular weight of 44.1kDa and pI of 5.53. Since the SECHI44 protein also appears to encode a signal peptide, an extracellular location for the corresponding protein is predicted. Comparison of SECHI44 sequence with known sequences of fungal endochitinases revealed that SECHI44 is grouped with endochitinases from other mycoparasites. Real-time quantitative RT-PCR analysis showed an elevated level of expression of sechi44 (21-fold) in chitin-rich (induced) as compared to no-carbon (non-induced) culture conditions. In dual culture, the temporal expression of sechi44 increased after 2 days of contact with R. solani, reaching a 10-fold increase after 9 days, followed by a decrease to basic expression level at 12 days. Interestingly, inhibition of sechi44 expression was observed when S. elegans hyphae were in close proximity with R. solani hyphae.     

The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli

Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3 end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.     

Immunogenic protein MPB57 from Mycobacterium bovis BCG: Molecular cloning, nucleotide sequence and expression

Using a single-probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an M_r of 10 818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.     

The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins

Summary An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.     

华北大黑鳃金龟中肠丝氨酸蛋白酶cDNA克隆、 序列分析及表达

丝氨酸蛋白酶是昆虫体内一类重要的消化酶, 为了了解该类酶的分子特性及功能, 本研究利用粉纹夜蛾Trichoplusia ni围食膜蛋白多克隆抗体筛选华北大黑鳃金龟Holotrichia oblita中肠cDNA表达文库, 首次得到编码华北大黑鳃金龟丝氨酸蛋白酶cDNA序列, 命名为HoSP1（GenBank登录号为FJ573146）。序列分析表明, 该基因长902 bp, 开放阅读框（ORF）长783 bp, 编码260个氨基酸, 推测分子量和pI值分别为26.7 kDa和4.19, 不含有N-糖基化位点, 但在Thr₁₅₇处有一个O-糖基化位点, 含有6个保守的半胱氨酸残基, 组成3对二硫键, 对于维持蛋白质的三级结构起着重要的作用。通过与几种丝氨酸蛋白酶的比对发现, 该酶具有组氨酸（His）、 天冬氨酸（Asp）、 丝氨酸（Ser）催化中心, 与褐新西兰肋翅鳃金龟Costelytra zealandica的14种丝氨酸蛋白酶有明显的相似性, 其中与CzSP3的序列一致性最高, 为52.47%。把该基因与pET21b载体重组后, 进行体外表达, 以BTEE为底物, 测得该酶的活力为0.0378 μmol/mg·min。HoSP1基因的克隆及体外表达为进一步研究该酶在华北大黑鳃金龟体内的表达及功能提供了依据。     

Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis.

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha-Galactosidase A from Pseudomonas fluorescens subsp. cellulosa: cloning, high level expression and its role in galactomannan hydrolysis

A library of Pseudomonas fluorescens subsp. cellulosa genomic DNA, constructed in lambda ZAPII, was screened for alpha-D-galactosidase activity. The DNA inserts from six galactosidase-positive clones were rescued into plasmids. Restriction digestion and Southern analysis revealed that each of the plasmids contained a common DNA sequence. The sequence of the Pseudomonas DNA in one of the plasmids revealed a single open reading frame (aga27A) of 1215 bp encoding a protein of M(r) 45900, designated alpha-galactosidase 27A (Aga27A). Aga27A exhibited extensive sequence identity with alpha-galactosidases in glycoside hydrolase 27, and appeared to be a single domain protein. The recombinant alpha-galactosidase was expressed at high levels in Escherichia coli and the biophysical properties and substrate specificity of the enzyme were evaluated. The data showed that Aga27A was a mesophilic neutral acting non-specific alpha-galactosidase. Both P. fluorescens subsp. cellulosa mannanase A (ManA) and Aga27A hydrolyse the polymeric substrate, carob galactomannan. Sequential hydrolysis with AgaA followed by ManA, or ManA followed by AgaA enhanced product release. The positive effects of sequential hydrolysis are discussed.     

梨小食心虫化学感受蛋白cDNA的克隆、序列分析及原核表达

为了研究梨小食心虫Grapholita molesta化学感受蛋白（chemosensory proteins, CSPs）在化学感受系统中的作用, 本研究利用RT-PCR和RACE技术克隆到一条梨小食心虫化学感受蛋白的全长cDNA序列, 命名为GmolCSP （GenBank 登录号： JQ821389）。序列分析表明, GmolCSP开放阅读框序列为384 bp, 编码127个氨基酸残基, 预测N末端含有18个氨基酸组成的信号肽序列, 其成熟蛋白的预测分子量为12.80 kD, 等电点为8.33。该基因编码的氨基酸序列与其他鳞翅目昆虫化学感受蛋白的氨基酸序列具有较高同源性。RT-PCR结果显示, GmolCSP在梨小食心虫成虫触角、 去触角的头、 胸、 腹、 足和翅中都有表达。将GmolCSP重组到表达载体pET-32a中, 转入大肠杆菌Escherichia coli BL21（DE3）进行表达。SDS-PAGE和Western 印迹检测结果显示, 梨小食心虫化学感受蛋白基因在大肠杆菌中成功地表达出一个分子量约为29 kD的融合蛋白, 与预测的融合蛋白分子量大小一致。本研究结果为进一步研究该蛋白的分子结构和功能奠定了良好基础。     

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

Molecular cloning,expression pattern and phylogenetic analysis of myosin light chain 2 gene from Antheraea pernyi: A potential marker for phylogenetic inference

Myosin light chain 2 (MLC-2) gene was isolated and characterized from Antheraea pernyi, a well-known wild silkmoth. The isolated cDNA sequence is 905 bp in length with an open reading frame of 612 bp encoding a polypeptide of 203 amino acids. Semi-quantitative RT-PCR analysis showed that the MLC-2 gene was transcribed during four developmental stages (egg, larva, pupa, and moth), and present in all tissues tested. Alignment analysis revealed that the deduced protein sequence has over 95% identity to myosin light chain 2 of lepidopteran species, and 57–88% identity to other insect species, suggesting that insect MLC-2 proteins are highly conserved throughout evolution. The protein sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate MLC-2 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of MLC-2 gene in phylogenetic study.     

Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: genes, expression patterns and protein interactions of two potential moonlighting proteins

Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.