scholarly journals Cloning, expression, purification of active recombinant human 12R-LOX enzyme and its inhibition by Acalypha indica extracts

2017 ◽  
Author(s):  
Naireen Fatima ◽  
Ramakrishna B P ◽  
Naresh Kumar ◽  
Pallu Reddanna
Keyword(s):  
Arachidonic Acid ◽  
Inflammatory Diseases ◽  
Expression System ◽  
Cost Effective ◽  
Single Step ◽  
Assay System ◽  
Traditional Medicines ◽  
Prokaryotic Expression System ◽  
Free Arachidonic Acid ◽  
Acalypha Indica

12R-LOX over-expression has been reported in various pathologies such as psoriasis, proliferative dermatitis as well as pulmonary obstructive diseases indicating that this enzyme plays significant role in pathogenesis of inflammatory diseases. 12R-LOX, therefore, is a suitable target for therapeutic intervention with potential application of its inhibitors in the treatment of skin and other inflammatory disorders. Identification of such inhibitors requires sufficient quantity of active enzyme to be produced by an easy and cost effective expression systems and further development of a robust assay system to screen inhibitors against the 12R-LOX enzyme. Therefore, in the present study, a prokaryotic expression system was developed to over-express and purify active human 12R-LOX enzyme by a single step purification process. We have further standardized an HPLC based assay system to assess the activity of purified human 12R-LOX enzyme. We show here that purified 12R-LOX preferentially utilizes free arachidonic acid as the substrate but it is also active on methyl ester of arachidonic acid, albeit less efficiently. Additionally, using this assay system we observed the potent inhibition of human 12R-LOX enzyme activity by the ethyl acetate and aqueous sub-fractions of Acalypha indica leaves, which is widely used in traditional medicines for the treatment of various skin ailments

scholarly journals Cloning, expression, purification of active recombinant human 12R-LOX enzyme and its inhibition by Acalypha indica extracts

2017 ◽  
Author(s):  
Naireen Fatima ◽  
Ramakrishna B P ◽  
Naresh Kumar ◽  
Pallu Reddanna
Keyword(s):  
Arachidonic Acid ◽  
Inflammatory Diseases ◽  
Expression System ◽  
Cost Effective ◽  
Single Step ◽  
Assay System ◽  
Traditional Medicines ◽  
Prokaryotic Expression System ◽  
Free Arachidonic Acid ◽  
Acalypha Indica

12R-LOX over-expression has been reported in various pathologies such as psoriasis, proliferative dermatitis as well as pulmonary obstructive diseases indicating that this enzyme plays significant role in pathogenesis of inflammatory diseases. 12R-LOX, therefore, is a suitable target for therapeutic intervention with potential application of its inhibitors in the treatment of skin and other inflammatory disorders. Identification of such inhibitors requires sufficient quantity of active enzyme to be produced by an easy and cost effective expression systems and further development of a robust assay system to screen inhibitors against the 12R-LOX enzyme. Therefore, in the present study, a prokaryotic expression system was developed to over-express and purify active human 12R-LOX enzyme by a single step purification process. We have further standardized an HPLC based assay system to assess the activity of purified human 12R-LOX enzyme. We show here that purified 12R-LOX preferentially utilizes free arachidonic acid as the substrate but it is also active on methyl ester of arachidonic acid, albeit less efficiently. Additionally, using this assay system we observed the potent inhibition of human 12R-LOX enzyme activity by the ethyl acetate and aqueous sub-fractions of Acalypha indica leaves, which is widely used in traditional medicines for the treatment of various skin ailments

scholarly journals Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System

2009 ◽  
Vol 75 (12) ◽  
pp. 3980-3986 ◽  
Author(s):  
Su A Jang ◽  
Bong Hyun Sung ◽  
Ju Hyun Cho ◽  
Sun Chang Kim
Keyword(s):  
Antimicrobial Peptides ◽  
Prokaryotic Expression ◽  
Inclusion Bodies ◽  
Expression System ◽  
Cost Effective ◽  
Exchange Chromatography ◽  
Insoluble Complex ◽  
Prokaryotic Expression System ◽  
Natural Forms ◽  
Cleavage Step

ABSTRACT We describe a novel prokaryotic expression system for the production of cationic antimicrobial peptides (AMPs). The method relies on a translationally coupled two-cistron system, in which the termination codon for the first cistron (which encodes the anionic polypeptide mIFc2, a derivative of human gamma interferon) overlaps with the initiation codon for the second cistron (which encodes a cationic AMP) in the sequence of 5′-TAATG-3′. By forming an insoluble complex with the AMP upon translation, the mIFc2 protein efficiently neutralized the toxicity of the coexpressed cationic AMP and minimized the sensitivity of AMP to proteolytic degradation in a host. The AMPs were retrieved from the insoluble inclusion bodies without any chemical or enzymatic cleavage step by simple cation-exchange chromatography. With our system, ∼100 mg of various AMPs (buforin IIb, parasin I, and pexiganan) were obtained from 1 liter of Escherichia coli culture. Our expression system may represent a universal cost-effective solution for the mass production of intact AMPs in their natural forms.

scholarly journals Expression and Purification along with Evaluation of Serological Response and Diagnostic Potential of Recombinant Sap2 Protein from C. parapsilosis for Use in Systemic Candidiasis

2021 ◽  
Vol 7 (12) ◽  
pp. 999
Author(s):  
Manisha Shukla ◽  
Pankaj Chandley ◽  
Harsimran Kaur ◽  
Anup K. Ghosh ◽  
Shivaprakash M. Rudramurthy ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Expression System ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Serological Response ◽  
Systemic Candidiasis ◽  
E Coli ◽  
Diagnostic Potential ◽  
Prokaryotic Expression System ◽  
Secreted Aspartyl Proteinase

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.

scholarly journals 38 Using pooled data for single-step genomic prediction: Impact of within-pool variance and size

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 9-9
Author(s):  
Johnna L Baller ◽  
Stephen D Kachman ◽  
Larry A Kuehn ◽  
Matthew L Spangler
Keyword(s):  
Phenotypic Variation ◽  
Equal Opportunity ◽  
Cost Effective ◽  
Single Step ◽  
Pool Size ◽  
Individual Data ◽  
Phenotypic Data ◽  
Breeding Values ◽  
Mean Values ◽  
Genetic Evaluations

Abstract Economically relevant traits (ERT) are routinely collected within commercial segments of the beef industry but are rarely included in genetic evaluations because of unknown pedigrees. Individual relationships could be resurrected with genomics, which would be costly; pooling DNA and phenotypic data provides a cost-effective solution. A simulated beef cattle population consisting of 15 generations was genotyped with approximately 50k markers (841 quantitative trait loci were located across the genome) and phenotyped for a moderately heritable trait. Individuals from generation 15 were included in pools (observed genotype and phenotype were mean values of a group). Estimated breeding values (EBV) were generated from a single-step GBLUP model. The effects of pooling strategy (random and minimizing or uniformly maximizing phenotypic variation), pool size (1, 2, 10, 20, 50, 100, or no data from generation 15), and generational gaps of genotyping on EBV accuracy (correlation of EBV with true breeding values) were quantified. Greatest EBV accuracies of sires and dams were observed when no gap between genotyped parents and pooled offspring occurred. The EBV accuracies resulting from pools were greater than no data from generation 15 regardless of sire or dam genotyping. Minimizing phenotypic variation increased EBV accuracy by 8% and 9% over random pooling and uniformly maximizing phenotypic variation, respectively. Pool size of 2 was the only scenario that did not significantly decrease EBV accuracy compared to individual data when pools were formed randomly or by uniformly maximizing phenotypic variation (P > 0.05). Pool sizes of 2, 10, 20, or 50 did not generally lead to EBV accuracies that were statistically different than individual data when pools were constructed to minimize phenotypic variation (P > 0.05). Pooled genotyping to garner commercial-level phenotypes for genetic evaluations seems plausible, although differences exist depending on pool size and pool formation strategy. The USDA is an equal opportunity employer.

Inside Cover: Implantation of Post-translational Tyrosylprotein Sulfation into a Prokaryotic Expression System (ChemBioChem 3/2011)

ChemBioChem ◽  
2011 ◽  
Vol 12 (3) ◽  
pp. 338-338
Author(s):  
Lu-Yi Lu ◽  
Bo-Han Chen ◽  
Jennifer Yun-Shin Wu ◽  
Chen-Chu Wang ◽  
Da-Huang Chen ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Expression System ◽  
Prokaryotic Expression System

scholarly journals Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Xiao Teng Ching ◽  
Yee Ling Lau ◽  
Mun Yik Fong ◽  
Veeranoot Nissapatorn ◽  
Hemah Andiappan
Keyword(s):  
Western Blot ◽  
Affinity Purification ◽  
Expression System ◽  
Economic Loss ◽  
Serum Samples ◽  
Serological Tests ◽  
Human Patient ◽  
Prokaryotic Expression System ◽  
Human Sera ◽  
Lysate Antigens

Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).

An efficient solution for the single-step synthesis of 4CaO·Al2O3·Fe2O3 powders

2009 ◽  
Vol 24 (1) ◽  
pp. 245-252 ◽  
Author(s):  
Robert Ianoş
Keyword(s):  
Combustion Synthesis ◽  
Solution Combustion Synthesis ◽  
Cost Effective ◽  
Fuel Mixture ◽  
Single Step ◽  
Combustion Reaction ◽  
Exothermic Effect ◽  
Solution Combustion ◽  
Time Saving ◽  
X Ray

Single-phase nanocrystalline 4CaO·Al2O3·Fe2O3 powders were prepared directly from the combustion reaction using a new cost-effective, time-saving, and environmentally friendly version of solution combustion synthesis. Instead of a single fuel, a fuel mixture of urea and β-alanine was used. It was shown by x-ray diffraction, energy-dispersive x-ray analysis, thermogravimetric analysis, and optical microscopy that this new version of the solution combustion synthesis allows the maximization of the exothermic effect associated with the combustion reaction. On the other hand, it was shown that the traditional version of combustion synthesis involving the use of a single fuel, such as urea or β-alanine, does not ensure the formation of Ca4Al2Fe2O10 unless subsequent thermal treatments are applied. It was suggested that the occurrence of combustion reactions cannot be regarded only in terms of adiabatic temperature, as the kinetic aspects overrule the thermodynamic ones.

scholarly journals Development of a Trivalent Construct Omp18/AhpC/FlgH Multi Epitope Peptide Vaccine Against Campylobacter jejuni

2022 ◽  
Vol 12 ◽  
Author(s):  
Hongqiang Lou ◽  
Xusheng Li ◽  
Xiusheng Sheng ◽  
Shuiqin Fang ◽  
Shaoye Wan ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Tandem Repeats ◽  
Expression System ◽  
Peptide Vaccine ◽  
Surface Protein ◽  
Infection Model ◽  
Protein Antigens ◽  
Epitope Peptide ◽  
Prokaryotic Expression System

Campylobacter jejuni (C. jejuni) is one of the major pathogens contributing to the enteritis in humans. Infection can lead to numerous complications, including but not limited to Guillain-Barre syndrome, reactive arthritis, and Reiter’s syndrome. Over the past two decades, joint efforts have been made toward developing a proper strategy of limiting the transmission of C. jejuni to humans. Nevertheless, except for biosecurity measures, no available vaccine has been developed so far. Judging from the research findings, Omp18, AhpC outer membrane protein, and FlgH flagellin subunits of C. jejuni could be adopted as surface protein antigens of C. jejuni for screening dominant epitope thanks to their strong antigenicity, expression of varying strains, and conservative sequence. In this study, bioinformatics technology was adopted to analyze the T-B antigenic epitopes of Omp18, AhpC, and FlgH in C. jejuni strain NCTC11168. Both ELISA and Western Blot methods were adopted to screen the dominant T-B combined epitope. GGS (GGCGGTAGC) sequence was adopted to connect the dominant T-B combined epitope peptides and to construct the prokaryotic expression system of tandem repeats of antigenic epitope peptides. The mouse infection model was adopted to assess the immunoprotective effect imposed by the trivalent T-B combined with antigen epitope peptide based on Omp18/AhpC/FlgH. In this study, a tandem epitope AhpC-2/Omp18-1/FlgH-1 was developed, which was composed of three epitopes and could effectively enhance the stability and antigenicity of the epitope while preserving its structure. The immunization of BALB/c mice with a tandem epitope could induce protective immunity accompanied by the generation of IgG2a antibody response through the in vitro synthesis of IFN-γ cytokines. Judging from the results of immune protection experiments, the colonization of C. jejuni declined to a significant extent, and it was expected that AhpC-2/Omp18-1/FlgH-1 could be adopted as a candidate antigen for genetic engineering vaccine of C. jejuni MAP.

Sign in / Sign up

Export Citation Format

Share Document