scholarly journals Cloning, Expression and Structural Modeling of the MlrA Protein from Novosphingobium sp. KKU25s for Microcystin Degradation

2021 ◽  
Vol 18 (15) ◽  
Author(s):  
Kranokpron MOOLWANG ◽  
Sakda DADUANG ◽  
Thidarat SOMDEE ◽  
Theerasak SOMDEE
Keyword(s):  
Nucleotide Sequence ◽  
Alpha Helix ◽  
Dimensional Structure ◽  
Base Pairs ◽  
3 Dimensional ◽  
Recombinant Cells ◽  
Pcr Product ◽  
Sds Page ◽  
Hplc Profile

MlrA is a gene involved in the degradation of toxic cyanobacterial microcystins. This gene encodes microcystinase, mlrA, the 1st enzyme in the pathway that breaks down toxic cyanobacterial microcystins. In this study, primers were designed, and polymerase chain reaction (PCR) was performed to amplify the mlrA gene in Novosphincgobium sp. KKU25s. A PCR product of 752 base pairs was obtained. The nucleotide sequence of the mlrA gene of Novosphingobium sp. KKU25s was similar to that of Sphingomonas sp. ACM-3962 (98 % similarity). The mlrA gene of Novosphingobium sp. KKU25s was further cloned into the pGEM T-Easy plasmid to obtain the nucleotide sequence of the mlrA gene. The gene was also ligated into the pET32a plasmid for gene expression. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and verified using SDS-PAGE. The expressed protein was approximately 22 kilodaltons. The cell-free extract (CE) containing the crude protein from confirmed recombinant cells showed high activity in the biodegradation of [Dha7] MC-LR. [Dha7] MC-LR at an initial concentration of 30 mg L-1 and was completely biodegraded within 30 h. A distinct product derived from [Dha7] MC-LR appeared with a decrease in the [Dha7] MC-LR peak in the HPLC profile. The product (m/z 999.51) showed a molecular weight of 18, which is higher than that of native [Dha7] MC-LR (m/z 981.50), and was determined to be a linearized peptide fragment of [Dha7] MC-LR using LC-MS analysis. The 3-dimensional structure of microcystinase was predicted from the amino acid sequence deduced from the mlrA gene by the Swiss Model and Phyre2 programs. The structure contained a predicted alpha helix. The predicted 3-dimensional structure was also similar to that of a protein in the CAAX protease group. HIGHLIGHTS Research focused on characterization of microcystinase (MlrA) protein First research worked on the degradation of [Dha7] MC-LR by MlrA This work is useful for the applications aimed at the removal of MCs in freshwater environments GRAPHICAL ABSTRACT

scholarly journals Streptomyces lavendulaeProtease Inhibitor: Purification, Gene Overexpression, and 3-Dimensional Structure

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
D. E. El-Hadedy ◽  
El-Sayed E. Mostafa ◽  
Moataza M. Saad
Keyword(s):  
Protease Inhibitors ◽  
Gel Filtration ◽  
Dimensional Structure ◽  
Fulminant Myocarditis ◽  
Ammonium Sulfate Precipitation ◽  
3 Dimensional ◽  
Pcr Product ◽  
Sds Page ◽  
Cvb3 Infection ◽  
Causative Agents

Protease inhibitorstrypsin (STI1, Streptomyces trypsin inhibitor 1) has been identified, purified by ammonium sulfate precipitation and Sephadex G-100 gel filtration. SDS-PAGE of protease inhibitor showed molecular weight of approximately 10 KDa. PCR product (~1615 bp) ofsti1gene was cloned in expression vectorpACYC177/ET3dand transformed inEscherichia coliJM109.Protease inhibitorstrypsin was purified and used as antivirus against Coxsackievirus B3 (CVB3). CVB3 is one of the major causative agents of chronic, subacute, acute, and fulminant myocarditis as well as pancreatitis and aseptic meningitis. It has been reported that more than 50% of human myocarditis is associated with CVB3 infection.

Preparation and characterization of (Ba,Sr)TiO3 thin films by liquid source chemical vapor deposition

2001 ◽  
Vol 672 ◽  
Author(s):  
Cheol-Hoon Yang ◽  
Young-Ki Han ◽  
Dong-Hyun Kim ◽  
Geun-Jo Han ◽  
Doo-Young Yang ◽  
...  
Keyword(s):  
Chemical Vapor ◽  
Precursor Solution ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Type Reactor ◽  
Step Coverage ◽  
Liquid Source ◽  
Bst Films ◽  
Gaseous Source

ABSTRACTThe cocktail source of BST was prepared by mixing of Ba, Sr, and Ti precursor solution with specific mole ratio. This cocktail source was vaporized and delivered into the warm wall reactor by liquid delivery system(LDS) and gaseous source was distributed by simple structure of gas injector instead of showerhead system. The thickness uniformity of BST on 8 inch wafers were less than 3%. The Ti composition uniformity of our films were less than the 1 at%(1σ ) at stoichiometric and near stoichiometric. Their dielectric constant was about 230 and leakage current density was lower than 10-8 A/cm2 under ±1V bias. Excellent step coverage and smooth (haze-free) surface morphology of BST films were obtained by a deposition using a noble dome type reactor. The merit of our warm wall type reactor also will be explained by excellent step coverage and the uniform composition with 3 dimensional structure. Our achievement should be applicable to the capacitor of next generation DRAM.

scholarly journals HOT PHENOL EXTRACTION OF TOTAL RNA FROM Thermoascus aurantiacus AND CHARACTERIZATION OF ITS THERMOSTABLE XYLANASE GENE

2016 ◽  
Vol 1 (1) ◽  
pp. 55-58
Author(s):  
Ang Chung Huap ◽  
Awang Ahmad Sallehin Awang Husaini ◽  
Hairul Azman Roslan
Keyword(s):  
Reverse Transcriptase ◽  
28S Rrna ◽  
Thermoascus Aurantiacus ◽  
Xylanase Gene ◽  
Base Pairs ◽  
Total Rna ◽  
Phenol Extraction ◽  
Pcr Product ◽  
Polymerase Chain

Total RNA was successfully isolated using hot phenol extraction method. Three bands representing the 18S, 5.8S and 28S rRNA was observed. No heavy smearing was observed in the RNA band patterns, indicating low levels of polysaccharide contamination, when subjected to 1% agarose gel electrophoresis. Genomic DNA was eliminated using DNase I digestion and lithium chloride (LiCl) precipitation. Two-steps reverse transcriptase polymerase chain reaction (RT-PCR) using M-MuLV Reverse Transcriptase and sequence specific primers for xylanase gene, XynA(F) and XynA(R), successfully generated the target amplicon of 500 base pairs (bp). Sequence analysis of the PCR product indicated as partial sequence of Thermoascus aurantiacus xylanase gene (XynA) deposited in the NCBI GenBank with accession number: AF127529.1 and AJ132635.1. Hot phenol extraction is useful for extracting large quantities of total RNA sufficient for complementary DNA (cDNA) synthesis in shorter period of time.

A structure-based approach to drug discovery; crystallography and implications for the development of antiparasite drugs

Parasitology ◽  
1997 ◽  
Vol 114 (7) ◽  
pp. 17-29 ◽  
Author(s):  
W. N. HUNTER
Keyword(s):  
Biological Activity ◽  
Dimensional Structure ◽  
Design Development ◽  
X Ray Diffraction ◽  
Physical Sciences ◽  
3 Dimensional ◽  
Large Molecules ◽  
Future Challenges ◽  
Recent Developments

Advances in the life and physical sciences have enabled us to characterize the 3-dimensional structure and the biochemical or biological activity of both small and large molecules. The use of structural chemistry to assist understanding of biological activity provides information relevant to the design, development or identification of new pharmaceuticals. This structure based approach has become an important component of drug research and is in widespread use by the major pharmaceutical companies. A brief historical introduction, to convey how this area of science has reached the present stage, is given. The basis of the structural approach to understanding the chemistry of small and large molecule biological activity is outlined with an emphasis on the use of results derived from X-ray diffraction methods. Developments in other areas are discussed to emphasize the multidisciplinary nature of this research and the benefits of combining different methods. Examples of protein crystallographic studies in the area of molecular parasitology, some of which are directly relevant to antiparasite drug design, are presented. The characterization of the enzyme trypanothione reductase, a project which has benefited from many of the recent developments, is detailed. Future challenges and difficulties, both scientific and economic, are discussed.

Metal/GaAs interfaces

1989 ◽  
Vol 47 ◽  
pp. 446-447
Author(s):  
C. Barry Carter ◽  
Jane G. Zhu ◽  
C. J. Palmstrøm
Keyword(s):  
Rare Earth ◽  
Earth Metal ◽  
Contact Layer ◽  
Dimensional Structure ◽  
Misfit Dislocations ◽  
Additional Advantage ◽  
3 Dimensional ◽  
Zinc Blende

Reactions between III-V compound semiconductors and metals occur during the formation of Ohmic or Schottky contacts for semiconductor devices. Since the products of such reactions usually have crystal structures different from the zinc-blende structure of GaAs, it is necessary to understand the structure of the metal/GaAs interface in order to optimize the quality of the contact. At low temperatures, ternary phases given of the form, MxGaAs, grow during the metal-GaAs reactions. Well-documented examples are given by Ni-GaAs and Co-GaAs reactions. These ternary phases are thermodynamically unstable and decompose to MyGa and MzAs at higher temperatures. For contacts involving rare-earth metals, such as Er, Tb and Dy, on GaAs, a substantial interface chemical reaction has been found in which GaAs is dissociated and a more than 12Å-thick rare-earth arsenide interfacial layer is formed. In order to improve the reliability of these contacts and to control the reactions which take place at the GaAs interface, intermetallic compounds, NiAl and CoGa, and rare-earth metal arsenides, YbAs and ErAs, have recently been grown epitactically on GaAs. The work described here has concentrated on the characterization of the compound/GaAs interfaces formed during this process, paying particular attention to the structure of the misfit dislocations present at the interface. An additional advantage of this approach is that, if the quality of the contact layer is sufficiently high, it might be possible to grow a second high-quality layer of GaAs on top and thus begin to generate a 3-dimensional structure.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

1988 ◽  
Vol 46 ◽  
pp. 152-153 ◽  
Author(s):  
A. Engel ◽  
A. Holzenburg ◽  
K. Stauffer ◽  
J. Rosenbusch ◽  
U. Aebi
Keyword(s):  
Membrane Proteins ◽  
Flow Rate ◽  
Lipid Membranes ◽  
Functional Characterization ◽  
Dimensional Structure ◽  
Electron Crystallography ◽  
Peltier Element ◽  
Protein Ratio ◽  
Precise Control ◽  
3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

Structure and Interaction of Protamine by Dark Field Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 160-161
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Field Electron ◽  
Proposed Model ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Positively Charged

It has been shown for some time that it is possible to obtain images of small unstained proteins, with a resolution of approximately 5Å using dark field electron microscopy (1,2). Applying this technique, we have observed a uniformity in size and shape of the 2-dimensional images of pure specimens of fish protamines (salmon, herring (clupeine, Y-l) and rainbow trout (Salmo irideus)). On the basis of these images, a model for the 3-dimensional structure of the fish protamines has been proposed (2).The known amino acid sequences of fish protamines show stretches of positively charged arginines, separated by regions of neutral amino acids (3). The proposed model for protamine structure (2) consists of an irregular, right-handed helix with the segments of adjacent arginines forming the loops of the coil.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

Sign in / Sign up

Export Citation Format

Share Document