scholarly journals Cloning, Amplified Expression and Bioinformatics Analysis of a Putative Nucleobase Cation Symporter-1 (NCS-1) Protein From Rhodococcus erythropolis

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Irshad Ahmad ◽  
Youri Lee ◽  
Nighat Nawaz ◽  
Rizwan Elahi ◽  
Israr Ali Khan ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Rhodococcus Erythropolis ◽  
Functional Studies ◽  
Grand Average ◽  
Sds Page ◽  
Secondary Transporter ◽  
Restriction Sites ◽  
Aliphatic Index ◽  
Pcr Technology ◽  
Nucleobase Cation Symporter 1

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3), then amplified expression of the DYC18_RS18060-His6 protein was achieved with detection by SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins. Copyright(c)  The Authors

A novel, fast-growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2539-2544 ◽  
Author(s):  
Ece S. Güner ◽  
Naoya Hashimoto ◽  
Teruki Kadosaka ◽  
Yasuyuki Imai ◽  
Toshiyuki Masuzawa
Keyword(s):  
Sequence Similarity ◽  
Optimal Growth ◽  
Hard Tick ◽  
The Novel ◽  
Borrelia Hermsii ◽  
Fast Growing ◽  
Sds Page ◽  
Northwestern Turkey ◽  
Observation Sequence ◽  
Pure Cultures

A novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium (family Ixodidae, subfamily Metastriata) using Barbour–Stoenner–Kelly (BSK) II medium. Tick samples were taken during the summer of 2000 from the Istanbul area in northwestern Turkey. Sixty-seven of 153 adults (44 %) and 72 of 185 nymphs (39 %) were infected with the novel spirochaete, whereas none of the 20 larvae examined were infected. The optimal growth temperature of the spirochaete in BSK II medium was 34–37 °C, and it could grow at 39 °C. Doubling times at 34 and 37 °C were 5·3 and 5·1 h, respectively. Six pure cultures of the spirochaete were obtained and characterized by microscopic observation, sequence analysis of the flagellin gene (flaB), SDS-PAGE and Western blotting. The spirochaete was morphologically similar to those of the genus Borrelia and contained a 41 kDa protein reactive with mAb H9724 specific to the flagellin of a Borrelia species. Polyclonal antibody raised to this spirochaete reacted with several antigen bands, whereas no bands were detected with Borrelia burgdorferi, Borrelia hermsii, Borrelia turicatae and Borrelia parkeri. The flaB sequences of the six isolates showed high similarity, with sequence similarity values ranging from 99·2 to 100 %; however, the similarity of the isolates' flaB sequences to those of the Lyme-disease-related Borrelia and relapsing-fever-associated Borrelia species was less than 90 %. These findings suggest that the unique spirochaete is a member of the genus Borrelia, and differs from previously described Borrelia species.

scholarly journals Designing of a novel multisubunit vaccine against Nipah virus structural proteins: A reverse vaccinology approach

2021 ◽  
Author(s):  
khalid Mohamed Adam
Keyword(s):  
Nipah Virus ◽  
Reverse Vaccinology ◽  
Structural Proteins ◽  
Effective Vaccine ◽  
Compact Structure ◽  
Grand Average ◽  
Physiochemical Parameters ◽  
Significant Public Health ◽  
Aliphatic Index ◽  
Stable Molecule

Abstract Background The significant public health risk posed by NiV zoonosis and the lack of effective countermeasures against the intermittent outbreaks of the disease in the South and Southeast Asia region have entailed an imperative search for a protective vaccine to prevent or mitigate its epidemic potentiality. This is an endeavor to design an effective, safe multisubunit vaccine using an in silico reverse vaccinology approach. Methods The epitopes used for the construction of the candidate vaccine were meticulously predicted from five viral structural proteins (G, F, M, N, P) using several immunoinformatics tools to assess different epitope characteristics, namely, VaxiJen server for antigenicity, IEDB immunogenicity tool for immunogenicity, AlgPred server for allergenicity, ToxinPred for toxigenicity, IFNepitope server for interferon-gamma induction, Protparam server for physicochemical properties, GROMACS for simulation and simulation dynamics analysis, and finally, SnapGene tool for molecular cloning. Results The proposed vaccine molecule consisted of 501 amino acids, encompassing 7 B cell epitopes, 14 CTL epitopes, and 4 HTL epitopes. The physiochemical parameters of the vaccine construct showed a molecular weight of 54.6 kDa, an acidic stable molecule with an instability index of 38.3, aliphatic index of 62.89, and grand average of hydropathicity of -0.476. Moreover, the docking results and simulation dynamics of the vaccine molecule and TLR-3 showed global energy of 1.58 Kcal/mol, atomic contact energy of 2.98 Kcal/mol, and RMSD of 0.65 nm. The radius gyration showed a relatively steady value throughout the simulation period. a suggestive result of a stable compact structure and a promisingly effective vaccine construct. Conclusion In summary, the overall results of the multi-subunit vaccine molecule are suggestive of a promisingly effective vaccine against NiV infection in humans with a relatively stable compact structure, however, further experimental validation and assessment of pathogenic priming and autoimmunity induction are recommended.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel Yu ◽  
Megan A Outram ◽  
Emma Creen ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

Rhodococcus spelaei sp. nov., isolated from a cave, and proposals that Rhodococcus biphenylivorans is a later synonym of Rhodococcus pyridinivorans, Rhodococcus qingshengii and Rhodococcus baikonurensis are later synonyms of Rhodococcus erythropolis, and Rhodococcus percolatus and Rhodococcus imtechensis are later synonyms of Rhodococcus opacus

2021 ◽  
Vol 71 (7) ◽  
Author(s):  
Soon Dong Lee ◽  
In Seop Kim
Keyword(s):  
Type Species ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
Rhodococcus Erythropolis ◽  
Rhodococcus Opacus ◽  
Rrna Gene ◽  
The Novel ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

Two novel actinobacterial strains, designated C9-5T and C3-43, were isolated from soil samples of a cave in Jeju Island, Republic of Korea, and subjected to taxonomic study by a polyphasic approach. The organisms exhibited a typical rod–coccus developmental cycle during growth and grew at 10–30 °C, pH 5–9 and 0–3 % (w/v) NaCl. In 92 single-copy core gene sequence analysis, strain C9-5T was loosely associated with Rhodococcus tukisamuensis , albeit sharing low 16S rRNA gene sequence similarity (97.4 %). A combination of morphological and chemotaxonomic characteristics supported assignment with the genus Rhodococcus . With respect to 16S rRNA gene sequence similarity, the novel isolates showed the highest identity to the type strain of Rhodococcus subtropicus (98.7 % sequence similarity), followed by Rhodococcus olei (98.5 %) and Rhodococcus pedocola (98.4 %).The average nucleotide identity and digital DNA–DNA hybridization values between strain C9-5T and members of the genus Rhodococcus were ≤81.5 and ≤37.1 %, respectively. A set of physiological and chemotaxonomic properties together with overall genomic relatedness differentiated the novel isolates from members of the genus Rhodococcus , for which the name Rhodococcus spelaei sp. nov. is proposed. The type strain is C9-5T (=KACC 19822T=DSM 107558T). Based on genome analysis performed here, it is also proposed that Rhodococcus biphenylivorans Su et al. 2015 is a later heterotypic synonym of Rhodococcus pyridinivorans Yoon et al. 2000, Rhodococcus qingshengii Xu et al. 2007 and Rhodococcus baikonurensis Li et al. 2004 are later heterotypic synonyms of Rhodococcus erythropolis (Gray and Thornton 1928) Goodfellow and Alderson 1979 (Approved Lists 1980), and Rhodococcus percolatus Briglia et al. 1996 and Rhodococcus imtechensis Ghosh et al. 2006 are later heterotypic synonyms of Rhodococcus opacus Klatte et al. 1995.

A novel p64-related Cl−channel: subcellular distribution and nephron segment-specific expression

1999 ◽  
Vol 276 (3) ◽  
pp. F398-F408 ◽  
Author(s):  
John C. Edwards
Keyword(s):  
Chloride Channels ◽  
Sequence Similarity ◽  
Fluid Phase ◽  
Human Kidney ◽  
Specific Expression ◽  
Proximal Tubule Cells ◽  
Sds Page ◽  
Nephron Segment ◽  
Related Proteins

Several closely related proteins that have been implicated as chloride channels of intracellular membranes have recently been described. We report here the molecular cloning and characterization of a new member of this family from human cells. On the basis of sequence similarity, we conclude that this new protein represents the human version of a previously described protein from rat brain named p64H1. The human version of p64H1 (huH1) is a 28.7-kDa protein that shows an apparent molecular mass of 31 kDa by SDS-PAGE. A single 4.5-kb message is detected on Northern blots and is present in all tissues probed. The protein is expressed in an intracellular vesicular pattern in Panc-1 cells that is distinct from the endoplasmic reticulum, fluid-phase endocytic, and transferrin-recycling compartments, but which does colocalize with caveolin. In human kidney, huH1 is highly expressed in a diffuse pattern in the apical domain of proximal tubule cells. huH1 is expressed less abundantly in a vesicular pattern in glomeruli and distal nephron.

The Thrombin Protecting Function of a Complement Inhibitor in Human Plasma

1979 ◽  
Author(s):  
E.R. Podack ◽  
J.G. Curd ◽  
J.H. Griffin ◽  
H.J. Müller-Eberherd
Keyword(s):  
Complex Formation ◽  
Antithrombin Iii ◽  
Membrane Attack Complex ◽  
Protein S ◽  
Thrombin Time ◽  
Two Dimensional ◽  
Complement Inhibitor ◽  
Functional Studies ◽  
Sds Page ◽  
At Iii

S-protein (S) is a newly discovered 80,000 MW plosma glycoprotein. It functions as an inhibitor of the membrane attack complex of complement. We now wish to report that S also functions as thrombin protecting factor in coagulation; S forms a reversible complex with thrombin which is more resistant to inactivation by antithrombin III (AT III) than thrombin alone. An S-thrombin complex and on S-throm-bin-AT III complex were formed in clotted plasma and with isolated proteins as demonstrated by two dimensional Immunoelectrophoresis. Functional studies measuring the esterolytic or clotting activity of thrombin showed that S in the presence and absence of heparin decreased the rate of inactivation of thrombin by AT III. Similar results were observed using plasma. For example, in the presence of 0.04 u/ml heparin and 1.6 u/ml thrombin, the thrombin time of plasma depleted in S was 150 sec. as opposed to 15 sec. when the plasma was reconstituted with purified S. That this effect of S was due to a decreased inactivation of thrombin by AT III was demonstrated directly by SDS-PAGE analysis of plasma containing 125l-thrombin. In the presence of S the rate of formation of the 95,000 dalton 125I-thrombin-AT III complex was markedly decreased compared to the rate of complex formation in the S-depleted plasma. These data suggest that S may modulate the interactions of thrombin and AT III.

A New Case Of Dysfibrinogenemia : Isolation Of The Abnormal, Unclottable Fibrinogen Population

1981 ◽  
Author(s):  
M Jandrot-Perrus ◽  
M H Aurousseau ◽  
F Josso
Keyword(s):  
Factor Xiii ◽  
Gel Filtration ◽  
Healthy Woman ◽  
Fibrin Clot ◽  
Elution Volume ◽  
Functional Studies ◽  
Sds Page ◽  
Normal Functional ◽  
A Chain ◽  
And Control

In a 81-year-old healthy woman, gross abnormalities of fibrin formation in routine tests led to the discovery of a dysfibrinogenemia.Abnormal and control fibrinogens were purified in parallel using precipitation by glycine (Kazal) ; final clottability was 95-98 % for the control and 50 % for the patient’s fibrinogen. Electrophoretic behaviour of the fibrinogen momecule, the three chains and the products of fibrin cross-linking by factor XIII a was normal. Functional studies gave the following results : (i) delayed coagulation by thrombin, Reptilase and Venacil with gross abnormalities of the clot; (ii) inhibition of coagulation of normal fibrinogen ; (iii) poor fibrin monomer aggregation (opacimetry) ; (iv) delayed fibrinogen proteolysis by plasmin (SDS-PAGE) . Release of fibrinopeptide A by thrombin was incomplete (RIA).Fibrinogen NH2-terminal residues were found normal, but the presence of ALA-residue in fibrin clot and in the supernatant showed that part of fibrinogen was not clotted, either copolymerized with fibrin or remaining in solution. Gel filtration of the supernatant showed the presence of both soluble complexes and fibrinogen characterized by the elution volume of the peak and NH2-terminal analysis. This fibrinogen population was unclottable by thrombin and inhibited clotting of normal fibrinogen.These preliminary results suggest the existence of a defect on the A-a chain of this abnormal fibrinogen which was called fibrinogen Bondy.

scholarly journals Rothia aeria sp. nov., Rhodococcus baikonurensis sp. nov. and Arthrobacter russicus sp. nov., isolated from air in the Russian space laboratory Mir

2004 ◽  
Vol 54 (3) ◽  
pp. 827-835 ◽  
Author(s):  
Ying Li ◽  
Yoshiaki Kawamura ◽  
Nagatoshi Fujiwara ◽  
Takashi Naka ◽  
Hongsheng Liu ◽  
...  
Keyword(s):  
16S Rdna ◽  
Sequence Similarity ◽  
Rhodococcus Erythropolis ◽  
Rdna Sequence ◽  
Taxonomic Study ◽  
16S Rdna Sequence ◽  
Gram Positive Bacteria ◽  
16S Rdna Sequence Analysis ◽  
Rothia Mucilaginosa ◽  
Established Species

Four Gram-positive bacteria, strains A1-17BT, A1-22T, A1-3T and A1-8, isolated from the air in the Russian space laboratory Mir, were subjected to a polyphasic taxonomic study. Phylogenetic analysis of the bacteria based on their 16S rDNA sequence showed that they belong to the genera Rothia (A1-17BT), Rhodococcus (A1-22T) and Arthrobacter (A1-3T and A1-8). Morphological, physiological, chemotaxonomic and genomic characteristics supported the assignments of these strains to these genera, but they could not be classified as any existing species within each respective genus. 16S rDNA similarity values between strain A1-17BT and its neighbours, Rothia dentocariosa genomovar II, Rothia dentocariosa, Rothia mucilaginosa and Rothia nasimurium, were respectively 99·8, 98·0, 96·4 and 95·4 %. Polyphasic taxonomic evidence indicated that strain A1-17BT should be categorized together with the unofficially named Rothia dentocariosa genomovar II, but clearly differentiated them from the established species of the genus Rothia. Strain A1-22T formed a coherent cluster with Rhodococcus erythropolis, Rhodococcus globerulus, Rhodococcus marinonascens and Rhodococcus percolatus in 16S rDNA sequence analysis, but DNA–DNA relatedness values were only 45·5, 35·3, 18·9 and 21·9 %. Strains A1-3T and A1-8 shared 99·9 % 16S rDNA sequence similarity, and strain A1-3T showed the highest level of 16S rDNA similarity, 96·6 %, to Arthrobacter polychromogenes. Contrasting biochemical characteristics were also identified. Finally, as a result of the polyphasic taxonomic study, three of the strains are proposed as type strains of novel species: Rothia aeria sp. nov. (A1-17BT=GTC 867T=JCM 11412T=DSM 14556T), Rhodococcus baikonurensis sp. nov. (A1-22T=GTC 1041T=JCM 11411T=DSM 44587T) and Arthrobacter russicus sp. nov. (A1-3T=GTC 863T=JCM 11414T=DSM 14555T).

Bioinformatics Analysis of Laccase (Lac1) in Pycnoporus cinnabarinus

2010 ◽  
Vol 121-122 ◽  
pp. 502-506
Author(s):  
He Li ◽  
Guo Ying Zhou ◽  
Huai Yun Zhang ◽  
Liang Guo
Keyword(s):  
Amino Acid ◽  
Manganese Peroxidase ◽  
Bioinformatics Analysis ◽  
Instability Index ◽  
Pycnoporus Cinnabarinus ◽  
Grand Average ◽  
Glycosylation Sites ◽  
The World ◽  
Aliphatic Index

Pycnoporus cinnabarinus is a plant pathogen. It is common in many areas and is widely distributed throughout the world. Laccases of are some of the few oxidoreductases commercialized as industrial catalysts. In the present study, some characters of the amino acid sequence of P.cinnabarinus laccase (Lac1) were predicted and analyzed with the tools of bioinformatics. These results showed that the protein was composed of 20 kinds of amino acid; the theoretical pI of manganese peroxidase was 4.81 and the theoretical molecular weight of manganese peroxidase was 56292.0 Da; total number of atoms was 7806; the extinction coefficient was 58120 (280 nm). The N-terminal of the sequence considered was M (Met) and the estimated half-life was 30 hours (mammalian reticulocytes, in vitro). The instability index (II) was computed to be 34.50; this classifies the protein as stable. Aliphatic index was 82.64. Grand average of hydropathicity (GRAVY) was -0.063. There were 8 glycosylation sites, a signal peptide and conserved domains.

scholarly journals Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system

1992 ◽  
Vol 281 (2) ◽  
pp. 545-551 ◽  
Author(s):  
L H Chang ◽  
J Y Fan ◽  
L F Liu ◽  
S P Tsai ◽  
M F Tam
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Relative Activity ◽  
Cloning And Expression ◽  
Glutathione S Transferase ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
A Subunit

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

Sign in / Sign up

Export Citation Format

Share Document