scholarly journals Rational Design and Evaluation of a Multiepitope Chimeric Fusion Protein with the Potential for Leprosy Diagnosis

2009 ◽  
Vol 17 (2) ◽  
pp. 298-303 ◽  
Author(s):  
Malcolm S. Duthie ◽  
Marah N. Hay ◽  
Cecile Z. Morales ◽  
Lauren Carter ◽  
Raodoh Mohamath ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Rational Design ◽  
Clinical Symptoms ◽  
Antibody Binding ◽  
Amino Acid Sequences ◽  
Production Costs ◽  
Fusion Construct ◽  
Control Programs ◽  
Good Ability ◽  
Multibacillary Leprosy

ABSTRACT Despite the reduction in the number of leprosy cases registered worldwide as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains stable in many countries. This indicates that Mycobacterium leprae, the causative agent of leprosy, is still being transmitted and that, without an earlier diagnosis, transmission will continue and infection will remain a health problem. The current means of diagnosis of leprosy is based on the appearance of clinical symptoms, which in many cases occur after significant and irreversible nerve damage has occurred. Our recent work identified several recombinant antigens that are specifically recognized by leprosy patients. The goal of the present study was to produce and validate the reactivity of a chimeric fusion protein that possesses the antibody binding properties of several of these proteins. The availability of such a chimeric fusion protein will simplify future test development and reduce production costs. We first identified the antibody binding regions within our top five antigen candidates by performing enzyme-linked immunosorbent assays with overlapping peptides representing the amino acid sequences of each protein. Having identified these regions, we generated a fusion construct of these components (protein advances diagnostic of leprosy [PADL]) and demonstrated that the PADL protein retains the antibody reactivity of the component antigens. PADL was able to complement a protein that we previously produced (the leprosy IDRI [Infectious Disease Research Institute] diagnostic 1 [LID-1] protein) to permit the improved diagnosis of multibacillary leprosy and that had a good ability to discriminate patients with multibacillary leprosy from control individuals. A serological diagnostic test consisting of these antigens could be applied within leprosy control programs to reduce transmission and to limit the appearance of leprosy-associated disabilities and stigmatizing deformities by directing treatment.

scholarly journals Use of Protein Antigens for Early Serological Diagnosis of Leprosy

2007 ◽  
Vol 14 (11) ◽  
pp. 1400-1408 ◽  
Author(s):  
Malcolm S. Duthie ◽  
Wakako Goto ◽  
Greg C. Ireton ◽  
Stephen T. Reece ◽  
Ludimila P. V. Cardoso ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Early Stage ◽  
Diagnostic Techniques ◽  
The Philippines ◽  
Immunoglobulin M ◽  
Protein Antigens ◽  
Fusion Construct ◽  
Central Brazil ◽  
Leprosy Patients ◽  
Multibacillary Leprosy

ABSTRACT Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

scholarly journals Computational-Driven Epitope Verification and Affinity Maturation of TLR4-Targeting Antibodies

2021 ◽  
Vol 22 (11) ◽  
pp. 5989
Author(s):  
Bilal Ahmad ◽  
Maria Batool ◽  
Moon Suk Kim ◽  
Sangdun Choi
Keyword(s):  
Rational Design ◽  
Structural Integrity ◽  
Critical Role ◽  
Autoimmune Encephalitis ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Computational Techniques ◽  
Multiple Strategies ◽  
Binding Epitope

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody–antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.

scholarly journals Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

1990 ◽  
Vol 171 (2) ◽  
pp. 565-570 ◽  
Author(s):  
K Ritter ◽  
H Brestrich ◽  
B Nellen ◽  
H Kratzin ◽  
H Eiffert ◽  
...  
Keyword(s):  
Infectious Mononucleosis ◽  
Triosephosphate Isomerase ◽  
Clinical Symptoms ◽  
Glycolytic Enzyme ◽  
Amino Acid Sequences ◽  
Cellular Proteins ◽  
51Cr Release ◽  
Terminal Amino Acid ◽  
Sds Page ◽  
Terminal Amino

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.

scholarly journals Main Structural Targets for Engineering Lipase Substrate Specificity

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 747
Author(s):  
Samah Hashim Albayati ◽  
Malihe Masomian ◽  
Siti Nor Hasmah Ishak ◽  
Mohd Shukuri bin Mohamad Ali ◽  
Adam Leow Thean ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Process Conditions

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

scholarly journals Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4

1993 ◽  
Vol 292 (1) ◽  
pp. 69-74 ◽  
Author(s):  
W Asmara ◽  
U Murdiyatmo ◽  
A J Baines ◽  
A T Bull ◽  
D J Hardman
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Protonated Form ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Pseudomonas Cepacia ◽  
Amino Acid Residues ◽  
Substrate Complex ◽  
Key Residues

The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

Characterization, chromosomal mapping, and expression of different ubiquitin fusion protein genes in tissues from control and heat-shocked maize seedlings

1996 ◽  
Vol 74 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Ling Liu ◽  
J. Roger H. Frappier ◽  
Karen d'Ailly ◽  
Burr G. Atkinson ◽  
Daniel S. Maillet ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Fusion Protein ◽  
Specific Protein ◽  
Amino Acid Sequences ◽  
Chromosomal Mapping ◽  
Repeat Units ◽  
Acid Protein ◽  
Specific Mrnas ◽  
Shock Proteins

Organisms possess at least two multigene families of ubiquitins: the polyubiquitins, with few to several repeat units, which encode a ubiquitin monomer, and the ubiquitin fusion (or extension) protein genes, which encode a single ubiquitin monomer and a specific protein. This report provides details about two ubiquitin fusion protein genes in maize referred to as MubG7 (uwo 1) and MubG10 (uwo 2). Each has one nearly identical ubiquitin coding unit fused without an intervening nucleotide to an unrelated, 237-nucleotide sequence that encodes for a 79 amino acid protein. The derived amino acid sequences of the two fusion proteins show that they differ by five amino acids (substitution by either a serine or threonine). MubG7 maps to chromosome 8L162 and MubG10 maps to chromosome 1L131. Analyses of the role(s) of these genes in response to heat shock (1 h at 42.5 °C) reveal that the level of these fusion protein mRNAs in the radicles or plumules from 2-day-old seedlings does not change; however, heat shock does cause a marked reduction in the accumulation of these same gene-specific mRNAs in the radicles and plumules of 5-day-old seedlings. These data confirm the suggestion from our earlier work that there is precise modulation, in a gene-specific manner, of the response to developmental as well as environmental signals.Key words: ubiquitin, ubiquitin extension (or fusion) protein, maize, heat shock, heat shock proteins, gene expression, chromosome map.

scholarly journals Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A

1991 ◽  
Vol 280 (1) ◽  
pp. 219-224 ◽  
Author(s):  
D Andersons ◽  
Å Engström ◽  
S Josephson ◽  
L Hansson ◽  
H Steiner
Keyword(s):  
Fusion Protein ◽  
Trichoplusia Ni ◽  
Expression System ◽  
Protein A ◽  
Eukaryotic Expression ◽  
Antibody Binding ◽  
Biologically Active ◽  
Fusion Partner ◽  
Synthetic Antibody ◽  
Cecropin A

A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy.

scholarly journals Computational approach for selection of epitope-based dengue vaccine targets

2018 ◽  
Vol 14 (4) ◽  
pp. 605-618
Author(s):  
Phuc Nguyen ◽  
Ly Le
Keyword(s):  
Amino Acids ◽  
T Cell ◽  
Dengue Virus ◽  
Rational Design ◽  
Protein Sequences ◽  
Computational Approach ◽  
Amino Acid Sequences ◽  
Universal Vaccine ◽  
E Protein

High antigenic variability in the envelope (E) protein of different virus strains has been a major obstacle in designing effective vaccines for Dengue virus (DENV). To maintain their biological function, some parts of viral proteins remain stable during evolution thus one possible approach to solve this problem is to recognize specific regions within different protein sequences of E that have the tendency to stay constant through evolution. These regions may possess some special attributes to become a vaccine candidate against dengue virus. In this study, a computational approach was utilized to identify and analyze highly conserved amino acid sequences of the DENV E protein. Sequences of 9 amino acids or more were specifically focused due to their immune-relevant as T-cell determinants. Different bioinformatics tools were responsible for revealing conserved regions in the DENV E protein and constructing the phylogenetic tree from the sequence database. The tools also predicted immunogenicity of the identified vaccine targets. Ultimately, two peptide regions of at least 9 amino acids were chosen due to their high conserved attribute in more than 95% of all collected DENV sequences. Moreover, both of them was found to be immune-relevant by their correspondence to known or putative HLA-restricted T cell determinants. The conserved attribute of these sequences through the entire analysis of this study supports their potential as candidates for further in vitro experiments for rational design a universal vaccine which has longer and broader impact.

scholarly journals First detection and molecular characterization of kobuvirus from a dog in Turkey

2019 ◽  
Vol 75 (05) ◽  
pp. 6248-2019
Author(s):  
ZEYNEP AKKUTAY-YOLDAR ◽  
TAYLAN KOÇ B. ◽  
ÇIĞDEM OĞUZOĞLU T.
Keyword(s):  
Molecular Characterization ◽  
Rectal Swab ◽  
Clinical Symptoms ◽  
Phylogenetic Analyses ◽  
Amino Acid Sequences ◽  
Coding Region ◽  
Gastrointestinal Infections ◽  
Protein Coding ◽  
Pcr Product ◽  
Domestic Carnivores

Canine kobuvirus (CaKVs) is a newly emerging virus detected in dogs in several countries. However, kobuvirus infection has not yet been described in domestic carnivores in Turkey. In this study, we tested blood and rectal swab samples to determine the presence of kobuvirus in a dog with clinical symptoms by reverse transcription-polymerase chain reaction (RT-PCR), using 3D (RNA polymerase) region primers of canine kobuviruses. To provide molecular characterization data, the Maximum Likelihood (ML) method was used for the phylogenetic analyses. The PCR product of the partial protein-coding region of the 3D protein gene from the rectal swab was amplified, purified, and sequenced. Phylogenetic analysis of amino acid sequences suggests that our CaKV strain was closely related to US-CaKVs,and placed on a monophyletic clade as a sister branch localized in the CaKV cluster. These results indicate that CaKV exists in dogs in Turkey. With a similarity of 94.2–96.1%, it is like other CaKVs. To our knowledge, this is the first report of CaKV infection of a dog by in Turkey. Further studies are needed to determine its role in dog gastrointestinal infections.

scholarly journals Justification of parameters mining and technological structure of modular mine sites in terms water disposal

2020 ◽  
Vol 174 ◽  
pp. 02013
Author(s):  
Roman Shishkov ◽  
Valery Fedorin
Keyword(s):  
Rational Design ◽  
New Technologies ◽  
Coal Industry ◽  
Production Costs ◽  
Design Stage ◽  
Wastewater Disposal ◽  
Technological Structure ◽  
Mine Workings ◽  
Mine Sites ◽  
Installed Capacity

The high cost of any traditional coal mines consists of many factors their operation, one which is basically drainage. In mines and sections Russia, on average, for each ton coal mined, more than 2 m3 water is pumped from mine workings to day surface. For this work, more than 10 thousand pumping units with a total installed capacity about 1 million kW are used at mining enterprises. Therefore, rational design and proper operation, in particular, drainage plants mining enterprises are important technical and economic tasks today and in future, which leads to increased production volumes and a transition to a new stage in development coal industry using new technologies. The application mining technology structure mine sites located on a coal mine is considered. The result is that at design stage it becomes possible to highlight use of water pumping measures only at stage preparatory mine workings, reducing production costs for equipment and its operation, volumetric work, mine workings for water collectors and allocation personnel from drainage site to more significant work . A comparative analysis of two options is given, in terms efficiency wastewater disposal, namely actual situation of a certain period at enterprise SUEK-Kuzbass JSC mine Taldinskaya Zapadnaya-2 and the use mine sites.

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