Data supporting Ni-NTA magnetic bead-based fluorescent protease assay using recombinant fusion protein substrates

Mótyán, János András; Miczi, Márió; Bozóki, Beáta; Tőzsér, József

doi:10.1016/j.dib.2018.03.031

Cited by 10 publications

(24 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The individual optimization of the assay may be limited only by the compatibility of the affinity beads with the applied conditions, reagents, and additives. In agreement with the manufacturer's protocol, we also found that the affinity binding of substrates to the Ni-NTA bead surfaces substantially weakens at pH ≤ 6.5 15 . Therefore, it is recommended to apply substrate blank samples parallel to the reaction samples, and the rate of spontaneous substrate dissociation needs to be considered during the evaluation of results.…”

Section: Discussionsupporting

confidence: 86%

“…ii) Termination of reaction samples. Another advantage of the method is that the enzymatic reaction can be terminated without the use of heat denaturation treatment or any potentially interfering chemical agents 15 . The termination can be carried out simply by separating the magnetic beads from the reaction mixture, using a conventional magnetic particle concentrator.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Bozóki

Mótyán

Miczi

et al. 2019

JoVE

Self Cite

View full text Add to dashboard Cite

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes. In this assay system, the applied substrates contain N-terminal hexahistidine (His 6 ) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

show abstract

Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Bozóki

Mótyán

Miczi

et al. 2019

JoVE

Self Cite

View full text Add to dashboard Cite

show abstract

“…Besides the synthetic oligonucleotides widely used in protease assays, fluorescent proteincontaining substrates were also used in activity measurements. The applied fluorimetric protease assay has been designed and tested previously on HIV-1 and TEV PRs [31][32][33], and we successfully adapted it for the investigation of Ty1 PR. The previously designed pDest-His 6 -MBP-mTurquoise2 expression vector [31][32][33] has been modified to contain the coding sequence of a (GGGGS) 4 linker.…”

Section: Discussionmentioning

confidence: 99%

“…Recombinant fluorescent substrates were expressed in E. coli BL21(DE3) cells as previously reported [31][32][33]. The His 6 -tagged fluorescent recombinant protein substrates were purified from the supernatant of the lysed cells by the addition of Ni-NTA magnetic agarose beads (Qiagen) and were incubated for 30 min while continuously shaking.…”

Section: Expression and Purification Of Fluorescent Substratesmentioning

confidence: 99%

“…Finally, the magnetic beads binding the His 6 -tagged fluorescent recombinant protein substrates were washed with the following Ty1 cleavage buffers: cleavage buffer A (10 mM PIPES, 75 mM NaCl, 0.25% Nonidet P-40, 5% glycerol, 75 mM KCl, 12.5 mM NaH 2 PO 4 , 61.25 mM sodium-glutamate, 12.5 mM MgSO 4 , 0.125 mM CaCl 2 , 0.05% Tween20, pH 7.0) or cleavage buffer B (50 mM MES, 100 mM Tris, 50 mM sodium-acetate, 150 mM NaCl, 75 mM KCl, 12.5 mM NaH 2 PO 4 , 61.25 mM sodium-glutamate, 12.5 mM MgSO 4 , 0.125 mM CaCl 2 , 0.05% Tween20, pH 8.0). The purified His 6 -tagged fluorescent recombinant protein substrates were used for proteolytic assays, based on the method described previously [31][32][33].…”

Section: Expression and Purification Of Fluorescent Substratesmentioning

confidence: 99%

See 1 more Smart Citation

Biochemical characterization of Ty1 retrotransposon protease

et al. 2020

Self Cite

View full text Add to dashboard Cite

Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae. The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e.g. the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae, untagged and His 6-tagged forms of Ty1 protease were expressed in E. coli. After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.

show abstract

Site‐directed mutagenesis improves the practical application of L‐glutamic acid decarboxylase inEscherichia coli

Liu

Zhang

et al. 2023

Engineering in Life Sciences

View full text Add to dashboard Cite

γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process.The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application: Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters K m and V max of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E.

show abstract

Data supporting Ni-NTA magnetic bead-based fluorescent protease assay using recombinant fusion protein substrates

Cited by 10 publications

References 4 publications

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Biochemical characterization of Ty1 retrotransposon protease

Site‐directed mutagenesis improves the practical application of L‐glutamic acid decarboxylase inEscherichia coli

Contact Info

Product

Resources

About