Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag

Ceylan, Hamid; Erdoğan, Orhan

doi:10.3906/biy-1602-83

Cited by 9 publications

(6 citation statements)

References 60 publications

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“…One way to potentially improve the activity retention is to control the orientation of the bound protein using site-specific conjugation. Many such strategies have been developed such as biotin–streptavidin conjugates ,, and bio-orthogonal reactions such as tetrazine ligation and azide-alkyne click chemistry. , These strategies could prove difficult for use in AChE however, as they require mutations to the protein and recombinant AChE expression is difficult . It is important to note, however, that despite the dramatic reduction in the enzyme’s activity, this AuNP–AChE construct can still be useful for different applications.…”

Section: Resultsmentioning

confidence: 99%

“…55,56 These strategies could prove difficult for use in AChE however, as they require mutations to the protein and recombinant AChE expression is difficult. 57 It is important to note, however, that despite the dramatic reduction in the enzyme's activity, this AuNP−AChE construct can still be useful for different applications. For example, AChE is inhibited by organo- phosphates common in pesticides and chemical nerve agents and thus loses activity in the presence of these compounds.…”

Section: ■ Introductionmentioning

confidence: 99%

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Gold Nanoparticles Are an Immobilization Platform for Active and Stable Acetylcholinesterase: Demonstration of a General Surface Protein Functionalization Strategy

Handali

Webb

2022

ACS Appl. Bio Mater.

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Immobilizing enzymes onto abiological surfaces is a key step for developing protein-based technologies that can be useful for applications such as biosensors and biofuel cells. A central impediment for the advancement of this effort is a lack of generalizable strategies for functionalizing surfaces with proteins in ways that prevent unfolding, aggregation, and uncontrolled binding, requiring surface chemistries to be developed for each surface−enzyme pair of interest. In this work, we demonstrate a significant advancement toward addressing this problem using a gold nanoparticle (AuNP) as an initial scaffold for the chemical bonding of the enzyme acetylcholinesterase (AChE), forming the conjugate AuNP−AChE. This can then be placed onto chemically and structurally distinct surfaces (e.g., metals, semiconductors, plastics, etc.), thereby bypassing the need to develop surface functionalization strategies for every substrate or condition of interest. Carbodiimide crosslinker chemistry was used to bind surface lysine residues in AChE to AuNPs functionalized with ligands containing carboxylic acid tails. Using amino acid analysis, we found that on average, 3.3 ± 0.1 AChE proteins were bound per 5.22 ± 1.25 nm AuNP. We used circular dichroism spectroscopy to measure the structure of the bound protein and determined that it remained essentially unchanged after binding. Finally, we performed Michaelis−Menten kinetics to determine that the enzyme retained 18.2 ± 2.0% of its activity and maintained that activity over a period of at least three weeks after conjugation to AuNPs. We hypothesize that structural changes to the peripheral active site of AChE are responsible for the differences in activity of bound AChE and unbound AChE. This work is a proof-of-concept demonstration of a generalizable method for placing proteins onto chemically and structurally diverse substrates and materials without the need for surface functionalization strategies.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Gold Nanoparticles Are an Immobilization Platform for Active and Stable Acetylcholinesterase: Demonstration of a General Surface Protein Functionalization Strategy

Handali

Webb

2022

ACS Appl. Bio Mater.

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“…[65,66] If the activity of the AChE enzyme is reduced or stopped, acetylcholine molecules will be less degraded and thus the acetylcholine concentration will remain high and the solution for the disease may be present. [7,67] Preferred AChE inhibitors for treating AD are inhibitors that increase the activity of cholinergic neurotransmission by increasing the amount of ACh. [68,69] As the most potent inhibitor, the long-acting tacrine (IC 50 : 8.18 nM; K i : 8.59 ± 5.38 nM) compound has a hepatotoxic effect.…”

Section: Resultsmentioning

confidence: 99%

The inhibition profile of sesamol against α-glycosidase and acetylcholinesterase enzymes

Topal

2019

International Journal of Food Properties

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Sesamol (1,3-benzodioxol-5-ol) is found in the oilseed product, sesame. In our study, the inhibitory effects of sesamol compound on acetylcholinesterase and α-glycosidase enzymes were evaluated. Another aim of this study was to compare the inhibitory effects of sesamol to acarbose for α-glycosidase enzyme and tacrine for acetylcholinesterase (AChE). Sesamol exhibited noncompetitive inhibition for both metabolic enzymes. IC 50 values for AChE and αglycosidase enzymes were calculated as 86.63 nM and 99.00 nM, respectively. On the other hand, K i values were calculated as 46.68 nM and 75.33 nM for AChE and α-glycosidase enzymes, respectively. According to the obtained results showed effective inhibition at low concentrations. ARTICLE HISTORY

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“…The addition of molecular weight in both types of protein is due to the addition of protein tags from pET SUMO plasmids of 12-kDa. The expression of recombinant proteins fused with SUMO (small ubiquitinrelated modifiers) shows a significant increase of proteins in the yield whose expression is very difficult to find in E. coli (Lee, 2008;Gopal and Kumar, 2013;Ceylan and Erdogan, 2017). Western blotting on PVDF paper using a monoclonal anti-histidine tag antibody was done to ensure the SDS-PAGE results were similar with the expected proteins.…”

Section: Discussionmentioning

confidence: 99%

Cloning, Expression and T Cell Epitope Prediction of Fbpa and FBPB Genes of Mycobacterium Tuberculosis Clinical Isolates

Fihiruddin¹,

Artama²,

Wibawa³

et al. 2021

J microb biotech food sci

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The effective treatment and accurate diagnosis of tuberculosis (TB) are not established yet. The Bacillus Chalmette-Guerin vaccine did not provide significant results in the prevention of TB and had only 0-80% efficacy. The fbpA and fbpB genes of M. tuberculosis are antigenic proteins and considered to be virulence factors. They are capable of stimulating immune responses in TB patients. In this study, we observed cloning, expression and T-cell epitope prediction of fbpA and fbpB genes from clinical isolates. The isolates of MultiDrug-Resistant (MDR-TB) were cultured and extracted. The fresh Polymerase Chain Reaction (PCR) products of the fbpA and fbpB genes were inserted into pET SUMO plasmids and transformed into Escherichia coli BL21 (DE3), then expressed in LB medium induced by 1.0 μM of IPTG. Sample sequences were analyzed by ClustalW and NCBI BLAST programs. The T-cell epitope prediction was analyzed by GENETYX vers 8.0. The PCR results were 1071 bp (fbpA gene) and 978 bp (fbpB gene). The SDS-PAGE and Western blotting weighed 48-kDa (fbpA gene) and 46-kDa (fbpB gene). We obtained seven specific T-cell epitopes based on IAd Pattern Position on both genes. Based on Rothbard/Taylor Pattern Position, we discovered twenty-three and sixteen specific T-cell epitopes for fbpA and fbpB genes, respectively. The fbpA and fbpB genes that encode Ag85A and Ag85B proteins have epitopes that are recognized by lymphocyte T-cells and are potentially subunit TB vaccine candidates in the future.

show abstract

Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag

Cited by 9 publications

References 60 publications

Gold Nanoparticles Are an Immobilization Platform for Active and Stable Acetylcholinesterase: Demonstration of a General Surface Protein Functionalization Strategy

Gold Nanoparticles Are an Immobilization Platform for Active and Stable Acetylcholinesterase: Demonstration of a General Surface Protein Functionalization Strategy

The inhibition profile of sesamol against α-glycosidase and acetylcholinesterase enzymes

Cloning, Expression and T Cell Epitope Prediction of Fbpa and FBPB Genes of Mycobacterium Tuberculosis Clinical Isolates

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