Cloning,sequencing, and heterologous expression of a cellobiohydrolase cDNA from the basidiomycete Corticium rolfsii

From a Corticium rolfsii cDNA library, a clone homologous to other fungal cellobiohydrolase (CBH1) genes was isolated using the polymerase chain reaction. In the nucleotide sequence, one 1.6 kb long open reading frame coding for a polypeptide of 530 amino acid residues was detected which showed 64% identity with CBH1 of Phanerochaete chrysosporium. With expression of the 1.8 kb cDNA using the Aspergillus oryzae expression system, we detected microcrystalline cellulose (Avicel) hydrolyzing activity in the culture supernatant. The secreted protein, accompanied by the activity, was 89 kDa by SDS-polyacrylamide gel electrophoresis.     

Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria.

L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.     

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

中国红豆杉细胞色素P450还原酶的基因克隆、表达与活性分析

细胞色素P450还原酶(Cytochrome P450 reductase,CPR)是细胞色素P450羟基化酶电子传递链的组成部分,在生物体内起着重要的电子传递作用。文章从中国红豆杉(Taxuswallichiana var. Chinensis)愈伤组织细胞中克隆CPR基因(TchCPR),TchCPR含有一个2154bp碱基的阅读框,编码717个氨基酸残基;在氨基酸水平上它与裸子植物细胞色素P450还原酶的同源性(82%)高于其他被子植物的细胞色素P450还原酶(74%)。在大肠杆菌BL21(DE3)中诱导表达了全长和从N-端截短不同数目氨基酸残基的6个融合肽段,经亲和层析纯化,分析了表达的不同长度融合蛋白的电子传递效率。结果表明截短长度大于61个氨基酸残基肽段的胞色素P450还原酶都能够诱导表达,在表达水平上无显著差异,而截短61个氨基酸的CPR融合蛋白电子传递的催化活性(1.6057nmol Cyt Cred/min/μg TchCPR融合蛋白)高于其他4个融合蛋白。     

Isolation and characterization of MUC15, a novel cell membrane-associated mucin.

The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino-acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full-length cDNA encoding a protein of 330 amino-acid residues. N-terminal amino-acid sequencing of derived peptides and the purified protein confirmed 76% of the sequence and demonstrated presence of a cleavable signal peptide of 23 residues, leaving a mature protein of 307 amino acids. Database searches showed no homology to any other proteins. A survey of the human genome indicated the presence of a corresponding gene on chromosome band 11p14.3. Isolation and sequencing of the complete cDNA sequence of the human homologue proved the existence of the gene product (334 amino-acid residues). This novel mucin-like protein was named MUC15 by appointment of the HUGO Gene Nomenclature Committee. The deduced amino-acid sequences of human and bovine MUC15 demonstrated structural hallmarks characteristic for other membrane-bound mucins, such as a serine, threonine, and proline-rich extracellular region with several potential glycosylation sites, a putative transmembrane domain, and a short cytoplasmic C-terminal. We have shown the presence of O-glycosylations, identified N-glycosylations at 11 of 15 potential sites in bovine MUC15, and a splice variant encoding a short secreted mucin. Finally, analysis of human and bovine cDNA panels and libraries showed MUC15 gene expression in adult human spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone marrow, lymph node, tonsil, breast, fetal liver, bovine lymph nodes and lungs of both species.     

cDNA cloning and deduced amino acid sequence of microvitellogenin, a female specific hemolymph and egg protein from the tobacco hornworm, Manduca sexta

Microvitellogenin is a female-specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly(A)+ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by using antiserum against microvitellogenin. Northern blot analysis of poly(A)+ RNA isolated from different life stages and sexes reveals that mRNA coding for microvitellogenin is only present in adult female fat body. Immunoprecipitation of the protein product translated from hybrid selected mRNA indicates that the cDNA clone is specific for microvitellogenin. The complete nucleotide sequence of the 834-base pair cDNA insert has been determined by the dideoxy chain termination method. The cDNA sequence predicts that microvitellogenin is a protein of 232 residues with a calculated molecular weight of 26,201. The cDNA also predicts an amino-terminal extension of 17 residues which are not present in the mature form. This sequence appears to be a signal peptide. A comparison of the translated amino acid sequence with the sequences in the National Biomedical Foundation protein library did not establish any sequence homology with other known proteins.     

Cloning, expression and characterisation of a recombinant triosephosphate isomerase from Taenia solium

We isolated and characterised the cDNA that encodes the glycolytic enzyme, triosephosphate isomerase from Taenia solium. A 450 bp DNA fragment was obtained by the polymerase chain reaction using a cDNA from larval stage as template and degenerate oligonucleotides designed from conserved polypeptide sequences from TPIs of several organisms. The fragment was used to screen a T. solium larval stage cDNA library. The isolated cDNA, encoding a protein of 250 amino acids shares 44.8-59.6% positional identity with other known TPIs, in which the catalytic enzyme residues were conserved. The complete coding sequence of the T. solium TPI cDNA was cloned into the expression vector pRSET and expressed as a fusion protein with an N-terminal tail of six histidine residues. The catalytic activity of the purified protein was similar to other TPI enzymes. Northern and Southern blot analysis suggest that in T. solium, single gene exists for triosephosphate isomerase and that the gene is expressed in all stages of the parasite.     

Cloning and functional expression of the mucrosobin protein, a beta-fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu).

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5'-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His(41), Asp(84) and Ser(179) in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific beta-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquire a functional snake venom protein with therapeutic potential.     

Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse,a Model of Krabbe's Disease

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.     

Cloning, functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase

In this paper we report the cloning and full sequencing of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA from Vitis vinifera L. (VV) leaves, an enzyme belonging to the polyamine biosynthetic pathway, which appears to play an important role in the regulation of plant growth and development. The presence of two overlapping ORFs (tiny ORF and small ORF) upstream of the main ORF is reported in the Vitis cDNA. When the Vitis SAMDC cDNA was expressed in yeast without the two upstream ORFs, the resulting activity was about 50 times higher than the activity obtained with the full cDNA. These results demonstrated the strong regulatory activity of the tiny and small ORFs. RT-PCR expression analysis showed evidence of a similar mRNA level in all the tissues tested, with the exception of the petioles. The VV SAMDC was also modelled using its homologues from Solanum tuberosum and Homo sapiens as template. The present work confirmed, for the first time in a woody plant of worldwide economic interest such as grapevine, the presence of a regulatory mechanism of SAMDC, enzyme that has a well-established importance in the modulation of plant growth and development.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.     

Cloning,Sequencing, Expression and Structural Investigation of Mnemiopsin from <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis>: An Attempt Toward Understanding Ca<Superscript>2+</Superscript>-Regulated Photoproteins

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl₂. The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.     

Demonstration of apolipoprotein CII in guinea pigs. Functional characteristics, cDNA sequence, and tissue expression

In contrast to plasma from other mammals, guinea pig plasma does not stimulate the activity of lipoprotein lipases in vitro. This had led previously to the conclusion that guinea pigs lack an analogue to apolipoprotein CII (apoCII). By adsorption of lipid-binding proteins to lipid droplets, thereby separating them from other plasma components, we could demonstrate apoCII-like activity in guinea pig plasma. On electrophoresis, the CII-like activity co-migrated with one isoform of guinea pig apolipoprotein CIII, identified by amino-terminal amino acid sequence determination (40 residues). By isoelectric focusing in a narrow pH gradient, the activating protein was separated sufficiently from the dominating apoCIII isoform to allow sequence determination of 8 residues from the amino terminus. Six of these were identical to corresponding residues in apoCII from dog and monkey. With the aid of a human apoCII cDNA probe we identified one cross-hybridizing mRNA species (approximately 600 nucleotides) on Northern blots of guinea pig liver. Three positive clones were isolated from a guinea pig liver cDNA library using the same cDNA probe. The nucleotide sequence showed extensive similarities to the previously known human, monkey, and canine sequences, but the signal peptide was 3 amino acid residues longer in the guinea pig protein, and there was a deletion of 4 residues in the putative lipid binding domain. Northern blot analyses indicated that guinea pig apoCII is mainly expressed in the liver with little or no contribution from the intestine.     

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.     

Characterization,subsite mapping and N-terminal sequence of miliin,a serine-protease isolated from the latex of Euphorbia milii

Miliin is a serine protease purified from the latex of Euphorbia milii. This work reports the effect of pH and temperature on the catalytic activity of miliin, using fluorescence resonance energy transfer (FRET) substrates. Miliin displayed the highest activity at pH 9 and 35 °C. Subsite mapping shows that subsites S₂ to S₂′ prefer uncharged residues. The S₂ subsite prefers hydrophobic aliphatic amino acids (Val, Pro and Ile) and defines the cleavage site. This work is the first one that reports subsite mapping of Euphorbiacea proteases. The N-terminal sequence showed higher similarity (40%) with the serine protease LIM9 isolated from Lilium. The presence of Tyr, Pro and Lys at positions 2, 5 and 10 respectively, were observed for most of the serine proteases used for comparison. The N-terminal sequence has striking differences with those reported previously for milin and eumiliin, other serine proteases isolated from the latex of E. milii.     

Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.     

Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus

A fat body-specific chitinase cDNA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus chitinase (AvChit1) is 1515 bp long with an open reading frame (ORF) of 431 amino acid residues. AvChit1 possesses the chitinase family 18 active site signature and one N-glycosylation site. The deduced amino acid sequence of AvChit1 cDNA showed 43% identity to both Glossina morsitans morsitans chitinase and a human chitotriosidase, and 30-40% to some insect chitinases which lack both the serine/threonine and chitin binding domains. Southern blot analysis of genomic DNA suggested the presence of AvChit1 gene as a single copy. Northern and Western blot analysis and enzyme activity assay showed the tissue-specific expression of AvChit1 in the A. ventricosus fat body. The AvChit1 cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AvChit1 showed activity in the chitinase enzyme assay using 0.1% glycol chitin as a substrate. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that AvChit1 is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity.