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1.
Keiser KC Brandt KS Silver GM Wisnewski N 《Archives of insect biochemistry and physiology》2002,50(4):191-206
cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis. 相似文献
2.
Silver GM Gaines PJ Hunter SW Maddux JD Thomas RE Wisnewski N 《Archives of insect biochemistry and physiology》2002,51(3):136-150
The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem. 相似文献
3.
Brandt KS Silver GM Becher AM Gaines PJ Maddux JD Jarvis EE Wisnewski N 《Archives of insect biochemistry and physiology》2004,55(4):200-214
Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells. 相似文献
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Park PJ Chung JY Byun HG Seog DH Kim SK 《Bioscience, biotechnology, and biochemistry》2002,66(10):2279-2282
A cDNA sequence encoding a pore-forming subunit of ATP-sensitive potassium channel (Kir6.2 gene) of the bullfrog, Rana catesbeiana Shaw, termed RcKir6.2, was isolated from a liver cDNA library. The cDNA contained a single open reading frame of 1,173 bp encoding 391 amino acids with a calculated molecular mass of 42.9 kDa, which has a structural motif (a GFG motif) of the putative pore-forming loop of Kir6.2. Analysis of its phlyogenetic position revealed that the RcKir6.2 is close to Kir6.2 of rabbits. The predicted amino acid sequence shared sequence identity with Kir6.2 of Homo sapiens, Cavia porcellus, Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus by 95.9, 95.6, 96.7, 96.7 and 99.7%, respectively. Expression of RcKir6.2 was detected in various tissues, including heart, kidney, liver, lung, spleen, and stomach of the bullfrog. 相似文献
6.
Pot RG Stoof J Nuijten PJ de Haan LA Loeffen P Kuipers EJ van Vliet AH Kusters JG 《FEMS immunology and medical microbiology》2007,50(2):273-279
Urease activity is vital for gastric colonization by Helicobacter species, such as the animal pathogen Helicobacter felis. Here it is demonstrated that H. felis expresses two independent, and distinct urease systems. H. felis isolate CS1 expressed two proteins of 67 and 70 kDa reacting with antibodies to H. pylori urease. The 67-kDa protein was identified as the UreB urease subunit, whereas the N-terminal amino acid sequence of the 70-kDa protein displayed 58% identity with the UreB protein and was tentatively named UreB2. The gene encoding the UreB2 protein was identified and located in a gene cluster named ureA2B2. Inactivation of ureB led to complete absence of urease activity, whereas inactivation of ureB2 resulted in decreased urease activity. Although the exact function of the UreA2B2 system is still unknown, it is conceivable that UreA2B2 may contribute to pathogenesis of H. felis infection through a yet unknown mechanism. 相似文献
7.
A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV). 相似文献
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A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins. 相似文献
10.
Julie Anderson Kirihara John P. Hunsperger Walter C. Mahoney Joachim W. Messing 《Molecular & general genetics : MGG》1988,211(3):477-484
Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome. 相似文献
11.
Inokuchi T Ikuzawa M Mineta T Yasumasu S Kobayashi K 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2003,136(4):647-655
A cDNA library was constructed from a poly(A)+ RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn139-Leu140-Thr141) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases. 相似文献
12.
Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA 总被引:1,自引:0,他引:1
Zheng W Wise ML Wyrick A Metz JG Yuan L Gerwick WH 《Archives of biochemistry and biophysics》2002,401(1):11-20
The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and pIs of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues. 相似文献
13.
M I Merkulova S G Andreeva T M Shuvaeva S V Novoselov I V Peshenko M F Bystrova V I Novoselov E E Fesenko V M Lipkin 《FEBS letters》1999,450(1-2):126-130
cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis. 相似文献
14.
Yamahara T Shiono T Suzuki T Tanaka K Takio S Sato K Yamazaki S Satoh T 《The Journal of biological chemistry》1999,274(47):33274-33278
A novel extracellular Mn-superoxide dismutase (SOD) was isolated from a moss, Barbula unguiculata. The SOD was a glycoprotein; the apparent molecular mass of its native form was 120 kDa, as estimated by gel filtration chromatography, and that of its monomer was 22,072 Da, as estimated by time of flight mass spectroscopy. The protein had manganese with a stoichiometry of 0.80 Mn/monomer. The cDNA clone for a gene encoding the extracellular Mn-SOD was isolated. Sequence analysis showed that it has a strong similarity to germin (oxalate oxidase) and germin-like proteins (GLPs) of several plant species and possesses all the characteristic features of members of the germin family. The clone encoding this extracellular Mn-SOD was therefore designated B. unguiculata GLP (BuGLP). BuGLP had no oxalate oxidase activity. In addition, the cDNA for a gene encoding the moss mitochondrial Mn-SOD was isolated. Its amino acid sequence had little similarity to that of BuGLP, even though a close similarity was observed among the mitochondrial Mn-SODs of various organisms. BuGLP was the first germin-like protein that was really demonstrated to be a metalloprotein with Mn-SOD activity but no oxalate oxidase activity. 相似文献
15.
Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase. 总被引:29,自引:3,他引:26
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cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance. 相似文献
16.
Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis. 总被引:8,自引:0,他引:8
M You X Xuan N Tsuji T Kamio I Igarashi H Nagasawa T Mikami K Fujisaki 《Insect biochemistry and molecular biology》2001,32(1):67-73
A cDNA expression library prepared from mRNA of Haemaphysalis longicornis (H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128-148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue. 相似文献
17.
E Schwoebel S Prasad T M Timmons R Cook H Kimura E M Niu P Cheung S Skinner S E Avery B Wilkins 《The Journal of biological chemistry》1991,266(11):7214-7219
A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein. 相似文献
18.
K Hayashi K Kanda F Kimizuka I Kato K Sobue 《Biochemical and biophysical research communications》1989,164(1):503-511
Recently, the two Mr forms of caldesmon (Mr's in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified. h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells. In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence. This sequence predicts a protein of 771 amino acids with a Mr of 88,743. The central portion of this sequence is composed of a 10-fold repeat of conserved amino acid sequence containing 13-15 amino acids. Further, a recombinant protein produced in Escherichia coli containing the full-length h-caldesmon cDNA has been characterized. Although the Mr of h-caldesmon predicted from amino acid sequence is 88,743, native and recombinant proteins show the same mol. wt. with 150kDa as measured by SDS-PAGE. This discrepancy may be due to the acidic amino acid-rich sequences at the N-terminal and central portions. A recombinant protein produced in E. coli possesses calmodulin-, F-actin- and tropomyosin-binding abilities in common with the native h-caldesmon. 相似文献
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J L Martin N R Pumford A C LaRosa B M Martin H M Gonzaga M A Beaven L R Pohl 《Biochemical and biophysical research communications》1991,178(2):679-685
When the inhalation anesthetic halothane was administered to rats, a 58 kDa protein in the liver became covalently labeled by the trifluoroacetyl chloride metabolite of halothane. The amino acid sequences of the N-terminal and of several internal peptide fragments of the protein were 99% homologous to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha. The purified trifluoroacetylated 58 kDa protein or native 58 kDa protein, however, did not have phosphatidylinositol-specific phospholipase C activity. We conclude that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha may encode for a microsomal protein of unknown function. 相似文献