scholarly journals Transcriptome-Based Identification of a Functional Fasciola hepatica Carboxylesterase B

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1454
Author(s):  
Yaretzi J. Pedroza-Gómez ◽  
Raquel Cossio-Bayugar ◽  
Hugo Aguilar-Díaz ◽  
Silvana Scarcella ◽  
Enrique Reynaud ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Dna Sequences ◽  
Bioinformatics Analysis ◽  
Fasciola Hepatica ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cellular Membrane ◽  
Reading Frame ◽  
Protein Mass ◽  
Gene Coding ◽  
Sds Page

Bioinformatics analysis of the complete transcriptome of Fasciola hepatica, identified a total of ten putative carboxylesterase transcripts, including a 3146 bp mRNA transcript coding a 2205 bp open reading frame that translates into a protein of 735 amino acids, resulting in a predicted protein mass of 83.5 kDa and a putative carboxylesterase B enzyme. The gene coding for this enzyme was found in two reported F. hepatica complete genomes stretching 23,230 bp, containing two exons of 1282 and 1864 bp, respectively, as well as a 20,084 bp intron between the exons. The enzymatic activity was experimentally assayed on F. hepatica protein extracts by SDS-PAGE zymograms using synthetic chromogenic substrates, confirming both the theoretical molecular weight and carboxylesterase enzymatic activity. Further bioinformatics predicted that this enzyme is an integral component of the cellular membrane that should be active as a 167 kDa homodimer complex and polyacrylamide gel electrophoresis (PAGE) zymograms experiments confirmed the analysis. Additional bioinformatics analysis showed that DNA sequences that code for this particular enzyme are highly conserved in other parasitic trematodes, although they are labeled hypothetical proteins.

scholarly journals The Largest Open Reading Frame (pks12) in the Mycobacterium tuberculosis Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis

2003 ◽  
Vol 71 (7) ◽  
pp. 3794-3801 ◽  
Author(s):  
Tatiana D. Sirakova ◽  
Vinod S. Dubey ◽  
Hwa-Jung Kim ◽  
Michael H. Cynamon ◽  
Pappachan E. Kolattukudy
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Open Reading Frame ◽  
Pcr Analysis ◽  
Reading Frame ◽  
Sds Page

ABSTRACT The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-14C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

Cloning and characterization of an alternative splicing transcript of the gene coding for human cytidine deaminase

2007 ◽  
Vol 85 (1) ◽  
pp. 96-102 ◽  
Author(s):  
Bianca Cristina Garcia Lisboa ◽  
Tamara da Rocha Machado ◽  
Daniel Carvalho Pimenta ◽  
Sang Won Han
Keyword(s):  
Alternative Splicing ◽  
Dna Sequences ◽  
Genomic Sequence ◽  
Cytidine Deaminase ◽  
Alternative Form ◽  
Nih3t3 Cells ◽  
Reading Frame ◽  
Gene Coding ◽  
Identification And Characterization

Human cytidine deaminase (HCD) catalyzes the deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. The genomic sequence of HCD is formed by 31 kb with 4 exons and several alternative splicing signals, but an alternative form of HCD has yet to be reported. Here we describe the cloning and characterization of a small form of HCD, HSCD, and it is likely to be a product of alternative splicing of HCD. The alignment of DNA sequences shows that the HSCD matches HCD in 2 parts, except for a deletion of 170 bp. Based on the HCD genome organization, exons 1 and 4 should be joined and all sequences of introns and exons 2 and 3 should be deleted by splicing. This alternative splicing shifted the translation of the reading frame from the point of splicing. The estimated molecular mass is 9.8 kDa, and this value was confirmed by Western blot and mass spectroscopy after expressing the gene fused with glutathionine-S-transferase in the pGEX vector. The deletion and shift of the reading frame caused a loss of HCD activity, which was confirmed by enzyme assay and also with NIH3T3 cells modified to express HSCD and challenged against cytosine arabinoside. In this work we describe the identification and characterization of HSCD, which is the product of alternative splicing of the HCD gene.

scholarly journals Nucleotide sequence of a novel arylesterase gene from Vibro mimicus and characterization of the enzyme expressed in Escherichia coli

1994 ◽  
Vol 298 (3) ◽  
pp. 675-680 ◽  
Author(s):  
J F Shaw ◽  
R C Chang ◽  
K H Chuang ◽  
Y T Yen ◽  
Y J Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cholesterol Acyltransferase ◽  
Reading Frame ◽  
Gene Coding ◽  
Step Procedure ◽  
Sds Page ◽  
Vibrio Mimicus ◽  
Hydrophobic Amino Acids ◽  
Cloned Gene

A gene coding for an arylesterase of Vibrio mimicus was cloned. Sequence determination reveals that the esterase gene has an open reading frame of 600 nucleotides which encodes a protein of M(r) 22,300. The deduced amino acid sequence contain a pentapeptide GDSLS (residues 27-31), which was also found in the phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Substitution of Ser-29 by alanine or cysteine in the cloned gene abolished the esterase activity in the tributyrin plate assay. On the other hand, the activity was not lost when Ser-31 was changed to alanine. The cloned gene was expressed in Escherichia coli, and the protein purified by a four-step procedure. The purified protein migrated on SDS/PAGE as a single band with an apparent M(r) of 22,100. This enzyme favoured the hydrolysis of several arylesters and was classified as an arylesterase (EC 3.1.1.2). N-Terminal analysis showed that Ser-20 was the first amino acid of the mature secreted protein, suggesting that the N-terminal 19 hydrophobic amino acids served as a signal peptide.

scholarly journals Comparison of the Fibronectin-Binding Protein FNE fromStreptococcus equi Subspecies equi with FNZ from S. equi Subspecies zooepidemicus Reveals a Major and Conserved Difference

2001 ◽  
Vol 69 (5) ◽  
pp. 3159-3163 ◽  
Author(s):  
Hans Lindmark ◽  
Martin Nilsson ◽  
Bengt Guss
Keyword(s):  
Dna Sequences ◽  
Stop Codon ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Binding Activity ◽  
Base Pairs ◽  
Reading Frame ◽  
A Cell

ABSTRACT The gene fnz from Streptococcus equisubspecies zooepidemicus encodes a cell surface protein that binds fibronectin (Fn). Fifty tested isolates of S. equi subspecies equi all contain DNA sequences with similarity to fnz. This work describes the cloning and sequencing of a gene, designated fne, with similarity tofnz from two S. equi subspeciesequi isolates. The DNA sequences were found to be identical in the two strains, and sequence comparison of the fne andfnz genes revealed only minor differences. However, one base deletion was found in the middle of the fne gene and eight base pairs downstream of the altered reading frame there is a stop codon. An Fn-binding protein was purified from the growth medium of a subspecies equi culture. Determination of the NH2-terminal amino acid sequence and molecular mass, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the purified protein is the gene product of the 5′-terminal half of fne. Fn-binding activity has earlier only been found in the COOH-terminal half of FNZ. By the use of a purified recombinant protein containing the NH2 half of FNZ, we provide here evidence that this half of the protein also harbors an Fn-binding domain.

scholarly journals Construction and Characterization of Haemophilus ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats

2000 ◽  
Vol 68 (6) ◽  
pp. 3352-3361 ◽  
Author(s):  
Melanie J. Filiatrault ◽  
Bradford W. Gibson ◽  
Birgit Schilling ◽  
Shuhua Sun ◽  
Robert S. Munson ◽  
...  
Keyword(s):  
Lipid A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Haemophilus Ducreyi ◽  
Ionization Mass ◽  
Wild Type ◽  
Reading Frame ◽  
Plasmid Library ◽  
Sds Page ◽  
Isogenic Mutants

ABSTRACT To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) ofEscherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.

Expression, purification and identification of gibberellin-induced cysteine-rich protein ofGymanadenia conopsea

2009 ◽  
Vol 6 (3) ◽  
pp. 271-276 ◽  
Author(s):  
Liu Yuan ◽  
Meng Guo-Quan ◽  
Zhou Jian-Ping ◽  
Zhang Teng ◽  
Feng Juan ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Reading Frame ◽  
Partial Cdna Sequence ◽  
Cysteine Rich Protein ◽  
Transmission Electron ◽  
Sds Page ◽  
Partial Cdna

AbstractPrimers bearing restriction enzyme sites forEcoR I andHind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of thegcgasagene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation intoEscherichia coliBL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.

scholarly journals Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon Sulfolobus solfataricus

2003 ◽  
Vol 185 (7) ◽  
pp. 2112-2121 ◽  
Author(s):  
M. Ben Potters ◽  
Barbara T. Solow ◽  
Kenneth M. Bischoff ◽  
David E. Graham ◽  
Brian H. Lower ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sulfolobus Solfataricus ◽  
Sodium Dodecyl ◽  
Open Reading Frame ◽  
Phosphoglycerate Mutase ◽  
Divalent Metal Ions ◽  
Reading Frame ◽  
Sds Page

ABSTRACT When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [γ-32P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of ∼46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co2+ or Mn2+. The recombinant protein underwent autophosphorylation when incubated with either [γ-32P]ATP or [γ-32P]GTP. The site of phosphorylation was identified as Ser59, which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the 32P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of 32P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.

scholarly journals Purification, Cloning, and Sequencing of a 3,5-Dichlorophenol Reductive Dehalogenase from Desulfitobacterium frappieri PCP-1

2004 ◽  
Vol 70 (8) ◽  
pp. 4532-4537 ◽  
Author(s):  
Jacinthe Thibodeau ◽  
Annie Gauthier ◽  
Marie Duguay ◽  
Richard Villemur ◽  
François Lépine ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Motifs ◽  
Reading Frame ◽  
Reductive Dehalogenase ◽  
Apparent Homogeneity ◽  
Gene Coding ◽  
Acid Protein ◽  
Desulfitobacterium Hafniense ◽  
Titanium Citrate

ABSTRACT A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent Km for 3,5-dicholorophenol was 49.3 ± 3.1 μM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

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