The construction of bifunctional fusion proteins consisting of MutS and GFP

Stanisławska-Sachadyn, Anna; Sachadyn, Paweł; Ihle, Karolina; Sydorczuk, Cezary; Wiejacha, K.; Kur, Józef

doi:10.1016/j.jbiotec.2005.08.009

Cited by 11 publications

(4 citation statements)

References 30 publications

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“…However, a weaker green fluorescence and a blue-shift in excitation peak were observed for the GFP:AspDH fusion protein. A similar decrease in green fluorescence and a red-shifted excitation peak have been also reported for MutS-GFP [30] and GFP-TGB1 [31] fusion proteins, respectively.…”

Section: In Vitro Characterization Of the Protein Constructsupporting

confidence: 83%

Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N‐terminal of aspartate dehydrogenase

Özyurt

Evran

Telefoncu

2013

Biotech and App Biochem

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We developed a fluorescent protein construct by genetically fusing green fluorescent protein (GFP) to aspartate dehydrogenase from Thermotoga maritima. The fusion protein was cloned, heterologously expressed in Escherichia coli cells, and purified by Ni-chelate affinity chromatography. It was then introduced into a measurement cuvette to monitor its fluorescence signal. Aspartate dehydrogenase functioned as the biorecognition element, and aspartate-induced conformational change was converted to a fluorescence signal by GFP. The recombinant protein responded to l-aspartate (l-Asp) linearly within the concentration range of 1-50 mM, and it was capable of giving a fluorescence signal in 1 Min. Although a linear response was also observed for l-Glu, the fluorescence signal was 2.7 times lower than that observed for l-Asp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l-Asp in vitro, and employment of aspartate dehydrogenase scaffold as a biorecognition element. A few genetically encoded amino-acid biosensors have been described in the literature, but to our knowledge, a protein has not been constructed solely for determination of l-Asp. Periplasmic ligand binding proteins offer high binding affinity in the micromolar range, and they are frequently used as biorecognition elements. Instead of choosing a periplasmic l-Asp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme.

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Section: In Vitro Characterization Of the Protein Constructsupporting

confidence: 83%

Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N‐terminal of aspartate dehydrogenase

Özyurt

Evran

Telefoncu

2013

Biotech and App Biochem

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“…These results could be explained because a big protein like C. freundii DHAK (over 120 kDa), may disturb the folding of FBPA (35 kDa), more over since FBPA is translated after DHAK. [38] The covalent union of both enzymes also allows that some of their properties are transferred to the fusion protein. Thus, the T m of the fused enzyme is almost identical to that of FBPA and higher than would be expected if the fusion did not exercise any effect.…”

Section: Resultsmentioning

confidence: 99%

Preparation and Characterization of a Bifunctional Aldolase/Kinase Enzyme: A More Efficient Biocatalyst for CC Bond Formation

Iturrate

Sánchez-Moreno

Oroz‐Guinea

et al. 2010

Chemistry A European J

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Abstract:A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening five amino acid linker. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structure of the bifuctional enzyme has been analysed by CD spectroscopy, as well as that of the parental enzymes, in order to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Since S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centres in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channelling.

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“…The method, proposed here, was used first in order to circumvent the problem of PCR contaminations occurring in the course of investigations on MutS protein (Sachadyn et al 2000 ; Stanisławska-Sachadyn et al 2005 ; Stanisławska-Sachadyn and Sachadyn 2005 ; Stanisławska-Sachadyn et al 2006 ; Stanisławska-Sachadyn et al 2003 ) where 69 bp amplicons were quantitated with real-time PCR in order to estimate the amounts of DNA bound by the protein. As mentioned above, all efforts, such as applying all good laboratory practices, replacement of PCR reagents and materials, and trying an alternative PCR master-mix, were ineffective to eradicate the persisting contaminations.…”

Section: Resultsmentioning

confidence: 99%

A simple modification of PCR thermal profile applied to evade persisting contamination

Banasik

Stanisławska-Sachadyn

Sachadyn

2016

J Appl Genetics

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The polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem of persisting PCR contamination is reported. The principle of this approach lies in shortening the steps of denaturation, annealing, and elongation in the PCR thermal cycle. The modification leads to the radical decline of false positive signals obtained for the no-template controls without affecting the detection of target PCR products. In the model experiments presented here, the signal of negative control was shifted by about ten cycles up above those for the examined samples so that it could be neglected. We do not recommend this solution in PCR diagnostics, where the sensitivity of detection is of the highest priority. However, the approach could be useful to pass by the problem of persisting contamination in quantitative PCR, where the range of quantitation is usually much above the limits of detection.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-015-0336-z) contains supplementary material, which is available to authorized users.

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The construction of bifunctional fusion proteins consisting of MutS and GFP

Cited by 11 publications

References 30 publications

Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N‐terminal of aspartate dehydrogenase

Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N‐terminal of aspartate dehydrogenase

Preparation and Characterization of a Bifunctional Aldolase/Kinase Enzyme: A More Efficient Biocatalyst for CC Bond Formation

A simple modification of PCR thermal profile applied to evade persisting contamination

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