1,721,086 research outputs found
Caspase-3-facilitated stoichiometric cleavage of a large recombinant polyprotein
No full textIn this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of
caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was
partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The
cleavage efficiency of the DEVD-based substrate was approximately 2.0 times higher than that of the
IETD substrate, in response to caspase-3. Based on this, 3 protein genes of interest were genetically
linked to each other by adding two proteolytic cleavage sequences, DEVD and IETD, for caspase-3.
Particularly, glutathione-S transferase (GST), maltose binding protein (MBP), and red fluorescent protein
(RFP) were chosen as model proteins due to the variation in their size. The expressed polyprotein
was purified by immobilized metal ion affinity chromatography (IMAC) via a hexa-histidine tag at
the C-terminal end, showing 93 kDa of a chimeric GST:MBP:RFP fusion protein. In response to caspase-
3, cleavage products, such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa),
were separated from a large precursor GST:MBP:RFP (93 kDa) via SDS-PAGE. The results obtained
from this study indicate that a multi-protein can be stoichiometrically produced from a large polyprotein
by using proteolytic recognition motifs, such as DEVD and IETD tetra-peptides, for caspase-3.open
Strategies in protein immobilization on a gold surface
No full textProtein immobilization on a gold surface plays an important role in the usefulness of
biosensors that utilize gold-coated surfaces such as surface plasmon resonance (SPR), quartz
crystal microbalance (QCM), etc. For developing high performance biosensors, it is necessarily
required that immobilized proteins must remain biologically active. Loss of protein activity
and maintenance of its stability on transducer surfaces is directly associated with the choice
of immobilization methods, affecting protein-protein interactions. During the past decade, a
variety of strategies have been extensively developed for the effective immobilization of
proteins in terms of the orientation, density, and stability of immobilized proteins on analytical
devices operating on different principles. In this review, recent advances and novel strategies
in protein immobilization technologies developed for biosensors are briefly discussed, thereby
providing an useful information for the selection of appropriate immobilization approach.open
An overview of techniques in enzyme immobilization
No full textImmobilized enzymes have become the subject of considerable interest due to their excellent functional properties such as reusability, cost-effectiveness, and optimality during the past decades. Enzyme immobilization technology is not only used in industrial processes, but also a component technology of products for medical diagnostics, therapy, food industry, bio energy, and biomaterial detection. In this review, new methods for enzyme immobilization are introduced, and the advantages and disadvantages of a variety of techniques in enzyme immobilization will be also discussed.
Applications of animal biosensors
No full textOdorous compounds perceived by humans or animal species produce a response in chemical- or electronic-based analytical detection systems (chemical sensors, electronic noses, gas chromatography, mass spectrometers, and so on). Animal noses can also produce a recognizable behavioral response in the animal, when exposed to those compounds. Recently, much attention has been paid to the use of animals for scent detection based on their behavioral responses, referred to as animal biosensors. So far, behavioral odor detection by animals has been applicable in some fields, such as forensic sciences, homeland security, or, more recently, cancer diagnostics. The major advantage of animal biosensors is that the animals can be conditioned rapidly and cost-effectively, offering benefits in terms of noninvasive detection and early diagnosis. Here, we review the applications of living biosensors as whole animal biosensors and discuss the main issues, approaches, and challenges.open
DNA data storage in Perl
No full textHere we report a simple and flexible method for DNA data storage based on Perl script. For this approach, the text data of the preamble of the “Universal Declaration of Human Rights” consisting of 2,046 words was encoded into the corresponding 8,148 base pairs of DNA using Perl-based encoding with a hash table. The encoded DNA sequences were then artificially synthesized for storage. The information DNA consisted of a total of 22 chemically synthesized DNA fragments with 400 nucleotides each, which were inserted into a cloning vector to multiply the plasmid DNA. The nucleotide integrity of the data-carrying DNA sequences were ensured under the accelerated aging conditions. Also, an erroneous nucleotide in the information DNA sequences was successfully corrected using the overlap extension PCR method. The stored DNA was read by sequencing, and the resulting DNA sequence information was successfully decoded to convert the DNA records back to the original document. Our results indicate that textual data can be stored in DNA using a simple, easy, and flexible Perl by running a script from the command line.
Nanobiotechnology, today and tomorrow
No full textNanobiotechnology, the interdisciplinary area at the crossroad of biotechnology and nanoscience, combines contributions from molecular and cell biology, chemisty, material science, and physics in an attempt to understand the behavior of nanobiomaterials, their development and applications. At present, nanobiotechnology is believed to hold great promise for improving health and prolonging life, faciliating biomarker discovery, molecular diagnostics, discovery of novel drugs and drug delivery, which are important basic components of biomedical science. In the recent trend of nanobiotechnology, this review is intended to provide a better understanding of nanobiotechnology in its applications and perspectives, separating this integration technology into three parts such as nanobiochip/sensor, nanobiomaterials, and nanobioanalysis in order to hopefully gain insights into why size matters, how nano-materials and -devices can be engineered.open
Self-assembled monolayer fabrication of cysteine-modified ferredoxin
No full textRhodobacter sphaeroides ferredoxin is a metalloprotein with ferric ion in its active site. Ferredoxin has redox property and it can transfer the electron. These molecules can be applied to the bioelectronics by fabricating them as a self-assembled bio-film. The significant key of film fabrication is the immobilization method of bio-molecule. In our previous works, it has been reported that metalloprotein film can be fabricated by using chemical linker material that have thiol-group to assemble it on gold substrate. However, the chemical linker can interfere with electron transfer because it is acted as an insulator of the system. So, we used recombinant protein with cysteine functional residue at the end of the protein which can be directly immobilized on the gold (Au) surface. It could be confirmed the immobilization of the protein and surface morphology of thin film by surface plasmon resonance (SPR) and scanning tunneling microscope (STM). These results show that cysteine-modified ferredoxin can be used for making high quality protein film, and applied to the fabrication of nano-scale bioelectronics.open
SPR imaging-based monitoring of caspase-3 activation
No full textThe activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.open
Forkhead-associated domains of the tobacco NtFHA1 transcription activator and the yeast Fhl1 forkhead transcription factor are functionally conserved
No full textNtFHA1 encodes a novel protein containing the forkhead-associated (FHA) domain and the acidic domain in Nicotiana tabacum. NtFHA1 functions as a transactivator and is targeted to the nucleus. The sequence of the FHA domain of NtFHA1 is significantly homologous to that of the Fhl1 forkhead transcription factor of yeast. FHL1 was previously identified as a suppressor of RNA polymerase III mutations, and the fhl1 deletion mutant exhibited severe growth defects and impaired rRNA processing. Ectopic expression of the FHA domain of NtFHA1 (but not its mutant form) resulted in severe growth retardation in yeast. Similarly, expression of Fhl1, its FHA domain, or chimeric Fhl1 containing the NtFHA1 FHA domain also inhibited yeast growth. Yeast cells overexpressing the FHA domains of NtFHA1 and Fhl1 contained lower levels of mature rRNAs and exhibited rRNA-processing defects, similar to the fhl1 null mutant. Chimeric Fhl1 (but not the mutant form with a small deletion in its FHA domain) fully complemented the growth and rRNA-processing defects of the fhl1 null mutant, demonstrating that the FHA domain of NtFHA1 can functionally substitute for the FHA domain of Fhl1. These results demonstrate that the FHA domains of NtFHA1 and Fhl1 are conserved in their structure and function and that the FHA domain of Fhl1 is critically involved in regulation of rRNA processing in yeast. NtFHA1 function in plants may be analogous to Fhl1 function in yeast.open
Molecular characteristics and differential expression of two nuclear factors containing the FHA domain in Arabidopsis
No full textThe forkhead-associated (FHA) domain identified in a wide variety of proteins is a small module that recognizes phosphothreonine epitopes on proteins. AtFHAq and AtFHA2 of Arabidopsis are homologs of tobacco NtFHA1 encoding a FHA domain-containing transcription activator. Previously, we showed that the FHA domain of NtFHA1 functionally substitutes for that of the Fhl1 forkhead transcription factor involved in rRNA processing in yeast, despite significant differences in their protein structures. In this study, we characterized AtFHA1 and AtFHA2 of Arabidopsis and analyzed their expression patterns. AtFHA1 and AtFHA2 contain an N-terminal FHA domain and a C-terminal acidic region, but lack any known DNA-binding motifs. Both proteins were targeted to the nucleus. GUS staining of transgenic Arabidopsis plants containing the promoter-GUS fusion gene demonstrated high expression of AtFHA2 in whole tissues throughout plant development. In contrast, AtFHA1 was temporally expressed at low levels in roots and vascular tissues of stems. Interestingly, AtFHA1 expression was significantly induced in whole plants in response to external stimuli. The observed differential expression of AtFHA1 and AtFHA2 implies that these two genes have distinct regulatory roles in Arabidopsis.open
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