56 research outputs found
Production and Purification of a Novel Anti-TNF-α Single Chain Fragment Variable Antibody
Purpose: TNF-α is an
inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies
are being used in clinic for the purpose of diagnosis and treatment due to
their high specificity. The objective of the current study was to express and
purify an anti-TNF-α scFv antibody identified by phage display technology.
Methods: The DNA coding sequence of the identified scFv was
cloned into pET28a vector and the corresponding protein was expressed as 6Ă—His
tagged using E.coli BL21 (DE3) pLysS expression system followed by
affinity purification on Ni-Sepharose affinity column.
Results: The J44 scFv antibody was cloned into the expression
vector and successfully expressed and purified. The purity of the scFv fraction
was confirmed using SDS-PAGE analysis. Western blotting technique was used to
detect expression of 6Ă—His tagged protein.
Conclusion: In the current study an anti-TNF-α scFv antibody
was successfully expressed in bacterial expression system and purified on
affinity column. The purified protein can be used in different in vitro
and in vivo experiments in order to elucidate its functionality
Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain
Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5�10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications. © 2019 The Author(s)
Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain
Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications
Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana
Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (~ 100 fold) with double-motif probe than the single-motif probe. The overall Kd values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were ~2.3±1 and 2.5±1 µM, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo- and hetero-dimerize
6D-QSAR for predicting biological activity of human aldose reductase inhibitors using quasar receptor surface modeling
Abstract The application of QSAR analysis dates back a half-century ago and is currently continuously employed in any rational drug design. The multi-dimensional QSAR modeling can be a promising tool for researchers to develop reliable predictive QSAR models for designing novel compounds. In the present work, we studied inhibitors of human aldose reductase (AR) to generate multi-dimensional QSAR models using 3D- and 6D-QSAR methods. For this purpose, Pentacle and Quasar’s programs were used to produce the QSAR models using corresponding dissociation constant (Kd) values. By inspecting the performance metrics of the generated models, we achieved similar results with comparable internal validation statistics. However, considering the externally validated values, 6D-QSAR models provide significantly better prediction of endpoint values. The obtained results suggest that the higher the dimension of the QSAR model, the higher the performance of the generated model. However, more studies are required to verify these outcomes
In Vivo Effect of RSH-12, a Novel Selective MMP-9 Inhibitor Peptide, in the Treatment of Experimentally Induced Dry Eye Model
Methods and algorithms for molecular docking-based drug design and discovery
The role of technology in the medical field has resulted in significant developments within the pharmaceutical industry. Computational approaches have emerged as a crucial method in further advancing drug design and development.Methods and Algorithms for Molecular Docking-Based Drug Design and Discovery presents emerging research on the application of computer-assisted design methods for drugs, emphasizing the benefits and improvements that molecular docking has caused within the pharmaceutical industry. Focusing on validation methods, search algorithms, and scoring functions, this book is a pivotal resource for professionals, researchers, students, and practitioners in the field of theoretical and computational chemistry.xix, 456 pages : illustrations ; 29 c
Identification of Novel Single Chain Fragment Variable Antibodies Against TNF-α Using Phage Display Technology
Purpose: Tumor
necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both
physiological and pathological pathways. Because of central role of TNF-α in
pathogenesis of inflammatory diseases, in
the current study, we aimed to identify novel scFv antibodies against TNF-α
using phage display technology.
Methods: Using
libraries composed of phagemid displaying scFv antibodies, four rounds of
biopanning against TNF-α were carried out, which led to identification of scFvs
capable of binding to TNF-α. The scFv antibody with appropriate binding
affinity towards TNF-α, was amplified and used in ELISA experiment.
Results: Titration of phage achieved from different
rounds of biopanning showed an enrichment of specific anti-TNF-α phages during
biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying
J48 scFv antibody.
Conclusion: The findings in the
current work revealed that the identified novel scFv antibody displayed at the
N-terminal of minor coat proteins of phagemid binds TNF-α with suitable
affinity. However, the soluble form of the antibody is needed to be produced
and evaluated in more details regarding its binding properties to TNF-α
Molecular modeling of histamine H3 receptor and QSAR studies on arylbenzofuran derived H3 antagonists
Histamine H3 receptors are presynaptic autoreceptors found in both central and peripheral nervous systems of many species. The central effects
of these receptors suggest a potential therapeutic role for their antagonists in treatment of several neurological disorders such as epilepsy,
schizophrenia, Alzheimer’s and Parkinson’s diseases. The purpose of this study was to identify the structural requirements for H3 antagonistic
activity via quantitative structure–activity relationship (QSAR) studies and receptor modeling/docking techniques. A combination of partial least
squares (PLS) and genetic algorithm (GA) was used in the QSAR approach to select the structural descriptors relevant to the receptor binding
affinity of a series of 58 H3 antagonists. The descriptors were selected out of a pool of >1000 descriptors calculated by DRAGON, Hyperchem and
ACD labs suite of programs. The resulting QSAR models for rat and human H3 binding affinities were validated using different strategies. QSAR
models generated in the current work suggested the role of charge transfer interactions in the ligand–receptor interaction verified using the
molecular modeling of the receptor and docking two antagonists to the binding site. The 3D model of human H3 receptor was built based on bovine
rhodopsin structure and evaluated by molecular dynamics (MD) simulation in a mixed water–vacuum–water environment. The results were
indicative of the stability of the model relating the observed structural changes during the MD simulation to the suggested ligand–receptor
interactions. The results of this investigation are expected to be useful in the process of design and development of new potent H3 receptor
antagonists
Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus
Purpose: Robust pharmaceutical and industrial enzymes from extremophile
microorganisms are main source of enzymes with tremendous stability under harsh
conditions which make them potential tools for commercial and biotechnological
applications.
Methods: The genome of a Gram-positive halo-thermotolerant Bacillus
sp. SL1, new isolate from Saline Lake, was investigated for the presence of
genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase
(CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase
(blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG),
glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde
dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus
sp. SL1.
Results: Based on the DNA sequence alignment results, six of
the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the
nucleotide level to the same genes of B. licheniformis 14580
demonstrating extensive organizational relationship between these two strains.
Despite high similarities between the B. licheniformis and Bacillus
sp. SL-1 genomes, there are minor differences in the sequences of some enzyme.
Approximately 30% of the enzyme sequences revealed more than 99% identity with some
variations in nucleotides leading to amino acid substitution in protein
sequences.
Conclusion: Molecular characterization
of this new isolate provides useful information regarding evolutionary
relationship between B. subtilis and
B. licheniformis species. Since, the most industrial processes are
often performed in harsh conditions, enzymes from such halo-thermotolerant
bacteria may provide economically and industrially appealing biocatalysts to be
used under specific physicochemical situations in medical, pharmaceutical,
chemical and other industries
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