107 research outputs found
K+ is an indispensable cofactor for GrpE stimulation of ATPase activity of DnaK·DnaJ complex from Thermus thermophilus
AbstractK+ is an indispensable cofactor for ATPase activity of eukaryotic cytosolic Hsp70 chaperone systems which lack a GrpE homolog. In the case of the bacterial Hsp70 (DnaK) system, GrpE, a nucleotide exchange factor, stimulates ATPase activity but little is known about the effect of K+. Here, we have cloned a grpE gene from a thermophile, Thermus thermophilus, and purified a homodimeric GrpE protein. Using proteins of this bacterium, we found that the GrpE stimulation of ATPase activity of DnaK·DnaJ complex was absolutely dependent on the presence of K+
Functional Expression and Characterization of Tetrachloroethene Dehalogenase From Geobacter sp.
Reductive dehalogenase (RDase) consists of two parts, RdhA and RdhB. RdhA is the catalytic subunit, harboring a cobalamin cofactor and two Fe–S clusters. RdhA is anchored to the cytoplasmic membrane via the membrane anchoring subunit, RdhB. There are many genes encoding RDases in the genome of organohalide-respiring bacteria, including Dehalococcoides spp. However, most genes have not been functionally characterized. Biochemical studies on RDases have been hampered by difficulties encountered in their expression and purification. In this study, we have expressed, purified and characterized RdhA of RDase for tetrachloroethene (PceA) from Geobacter sp. PceA was expressed as a fusion protein with a trigger factor tag in Escherichia coli. PceA was purified and denatured in aerobic condition. Subsequently, this protein was refolded in the presence of FeCl3, Na2S and cobalamin in anaerobic condition. The reconstituted PceA exhibited dechlorination ability for tetrachloroethene. UV-Vis spectroscopy has shown that it contains cobalamin and Fe-S clusters. Since this method requires anaerobic manipulation only in the reconstituting process and has a relatively high yield, it will enable further biochemical studies of RDases
THE EXPRESSION AND PURIFICATION OF OCTA-ARGININE APOPTIN AND ITS ABILITY TO KILL CANCER CELLS
Objective: In this research, chicken anemia virus apoptin optimized genetically for expression in Escherichia coli and also modified using (His)6 tag, (Arg)8 tag, and HlyA tag intended for purification needs, penetration enhancement, and secretion from bacterial host to the growth media.Methods: The modified apoptin gene was optimized using an Integrated DNA Technology (IDT). The gene (606 bp) then ordered and synthesized by Eurofins. The apoptin gene was expressed using E. coli BL21 CodonPlus as host, in cultivation temperature of 37 °C, and 25 °C and purified using Ni-NTA agarose beads. The addition of (His) 6 tag enabled the apoptin to be purified in only one step by using nickel column. The expression and purification data analyzed qualitatively as well as quantitatively using SDS-PAGE. MTT assay was used to identify the antitumor effect of octa arginine-apoptin to two kinds of cancer cells, cervix HeLa cancer cell and colon Widr cancer cell. The viability of cell was analyzed when the cell incubated in the variation concentration protein for 72 h.Results: The constructed apoptin gene were expressed in E. coli successfully. The MTT assay indicated that Octaarginin-Apoptin was able to induce apoptosis of HeLa and Widr cells lines in a dose-dependent manner. The recombinant apoptin without tagging with octa-arginine, have no ability to induce apoptosis of HeLa and Widr cells lines. Conclusion: This octa arginine-apoptin may in the future allow the development of a therapeutic protein that is able to kill cancer cells specifically
Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K
AbstractThiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, γCys131-SO2H. When the SCNase α, β and γ subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (αβγ)4, like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase(+P15K)) possessed 0.86 Co atom/αβγ trimer and exhibited 78% of the activity of native SCNase. SCNase(+P15K) showed a UV–Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase(+P15K) had the γCys131-SO2H modification. These results indicate that SCNase(+P15K) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion
Single-molecule detection of chaperonin dynamics through polarization rotation modulation of CdSe QD luminescence imaging
We report our recent trials examining the single-molecule three-dimensional (3D) detection of protein conformational dynamics at room temperature. Using molecular chaperones as model proteins and cadmium selenide (CdSe) semiconductor quantum dots (QDs) as nanometer-scale probes, we monitored the temporal evolution of ATP-induced conformation changes with a total internal reflection fluorescence (TIRF) microscopy imaging technique in buffer solutions. The two-dimensional (2D) degenerate nature of the emission dipoles of the QDs, due to the uniaxial wurtzite crystal structure, made it possible to capture the 3D orientation using a polarization modulation technique in real time. The temporal resolution was half the period of analyzer rotation. Although still insufficient, the obtained signals suggest possible 3D detection of specific motions, which supports the two-step conformational changes triggered by ATP attachment.18th International Conference on Dynamical Processes in Excited States of Solids (DPC2013), August 4-9,2013, Fuzhou, Chin
Cloning of DNA Polymerase I Geobacillus thermoleovorans SGAir0734 from a Batu Kuwung Hot Spring in Escherichia coli
Access
to biological engineering has become a critical point of modern science
development through polymerase chain reaction (PCR). One of the main components
in this process is DNA polymerase, which copies the main template DNA. However,
there is a lack of studies on the production of DNA polymerase from indigenous
thermophilic bacteria in Indonesia. To examine this process, DNA polymerase I
gene (DNA pol I) from Geobacillus thermoleovorans (isolated from Batu
Kuwung, Banten, Indonesia) was transformed into Escherichia coli. The
gene was cloned by the cut and ligation method using NcoI and BamHI restriction
enzymes, which were ligated with a pET23d vector. The recombinant gene was
overexpressed in E. coli
and identified by using SDS-PAGE of 10%
acrylamide gel, showing that the protein molecular weight was approximately
. This
study successfully amplified the gene of interest, indicated by a high local
similarity between the sequencing results and theoretical gene and positive
intercellular protein expression. The results confirm that the study
successfully cloned and synthesized recombinant DNA pol I of Geobacillus
thermoleovorans from Batu Kuwung, Serang, Banten
Immobilization of Cholesterol Oxidase in Chitosan Magnetite Material for Biosensor Application
Cholesterol oxidase, a bio-catalyst that can catabolize cholesterol, has
proven applications in medicine. Here, a support material was used to enhance
the characteristics of the enzyme. Magnetite (Fe3O4) is
widely used as an enzyme support; however, the interaction between the enzyme
and the support should be capped with another material, such as chitosan biopolymer-based
material. In this study, chitosan-magnetite materials were synthesized by
mixing both compounds and activating with glutaraldehyde. The materials were
then characterized by Fourier Transform Infrared (FTIR) Spectroscopy. The
enzyme kinetic parameters were studied by following the cholesterol oxidation
reaction using high-performance liquid chromatography (HPLC) and comparing the
results between the free and the immobilized enzyme. The substrate
concentration was 2.5 mg/mL. The effect of enzyme concentration was tested
using different concentrations of enzyme (0.5, 1, and 2 mg/mL) to determine the
best operating conditions. The best conditions for the oxidation reaction were
immobilized enzyme at a 2 mg/mL concentration. Enzyme immobilization significantly
decreased the optimum substrate concentration to 0.1 mg/mL
Formation of highly toxic soluble amyloid beta oligomers by the molecular chaperone prefoldin
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