Analytical and diagnostic methods utilizing shigella flexneri apyrase

A technology of Shigella flexneri apyrase and apyrase, applied in biochemical equipment and methods, analytical materials, biological material analysis, etc., can solve the problem of substrate specificity and batch The difference between them cannot achieve complete degradation, influence and other issues

Inactive Publication Date: 2017-08-18
APIRAYS BIOSCI AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, current apyrase for DNA sequencing applications suffers from the following problems: complete degradation cannot be achieved due to enzyme quality (NDP kinase contamination), substrate specificity, and batch-to-batch variation, which not only leads to decreased Peaks and non-linear peaks also affect long-strand DNA sequencing

Method used

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  • Analytical and diagnostic methods utilizing shigella flexneri apyrase
  • Analytical and diagnostic methods utilizing shigella flexneri apyrase
  • Analytical and diagnostic methods utilizing shigella flexneri apyrase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0171] Example 1: Production and colorimetric evaluation of recombinant S. flexneri apyrase (SFA)

[0172] SFA was prepared and purified as described in the "Materials and Methods" section. Purified enzyme fractions were assayed for the presence of bacterial-derived apyrase using a colorimetric assay. from figure 1 As can be seen, the colorimetric assay clearly shows a color difference that directly corresponds to the expression level of apyrase, revealing its cellular localization. In all cases, a clear color difference between the non-induced and induced cultures could be seen compared to the color of the blank sample.

[0173] from figure 2 As can be seen, there is a clear correlation between the colorimetric analysis results of the clones and the apyrase expression profiles and their localization. The enzyme rSFA was consistently overexpressed at 25 kDa in induced cultures, especially in whole cell lysates and soluble fractions, but not in membrane fractions, thereby ...

Embodiment 2

[0174] Example 2: Sequence analysis between rSFA and STA

[0175] Once protein expression was confirmed by gel electrophoresis and colorimetric analysis, the rSFA gene product was sequenced to determine the nucleotide sequence for comparison with the reference sequence WP_010921592.1 obtained from the NCBI database ( Figure 3A ). As can be seen from the figure, the sequence of the cloned oligonucleotide fragment (rSFA) containing the SFA of apyrase-6xhistidine (Apyrase-6xHis) and the expressed sequence (pBL21-Apyrase-6xHis) fall within the desired range (see Figure 3A ). from Figure 3B As can be seen, the rSFA differs from the reference SFA only at the N- and C-terminus.

example example 3

[0176] Example 3: Compared with STA, rSFA is more efficient and stable in ATP degradation

[0177] Measure the decay of luminescence produced by the firefly luciferase reaction ( Figure 4 ) illustrates the ATP-degrading activity between two apyrases (rSFA and STA). Compared to STA, rSFA exhibited significant removal of ATP and its analogs within ~10 minutes. In other experiments, from Figure 5 It can be seen that the luminescence increases when ATP is added in both cases, whereas the addition of apyrase leads to a decay of luminescence, which is caused by ATP depletion. Deliberate addition of extra ATP during the reaction showed a similar trend of ATP consumption. However, rSFA exhibited faster ATP depletion activity than commercially available STA. When ATP and apyrase preparations were diluted 10-fold, ATP consumption decreased accordingly.

[0178] Stability was checked by storing rSFA samples at 4°C, followed by freezing and freeze-thawing. A study was performed to...

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Abstract

A method, comprising the steps of providing a sample containing contaminating nucleoside diphosphates and / or nucleoside triphosphates, such as ATP and / or ATP analogues including deoxyribonucleoside triphosphates;reducing the amount of the contaminating nucleoside diphosphates and / or nucleoside triphosphates in the sample with an apyrase enzyme, wherein said apyrase enzyme is a Shigella flexneriapyrase; andperforming an analysis of the sample, wherein said analysis comprises an assay that would have been affected by the contaminating nucleoside diphosphates and / or nucleoside triphosphates had they not been reduced in the reduction step.

Description

technical field [0001] The present invention relates to methods for the analysis and diagnosis of contaminating nucleotides. In particular, the invention relates to the determination or quantification of the amount of ATP in samples which may contain contaminating nucleotides, as well as reagents for use in such methods and the production of such reagents. Background technique [0002] Adenosine triphosphate (ATP) is a molecule present in all living cells. Since the concentration of ATP in cells is fairly constant, measuring the amount of ATP in a sample can be used as an alternative method to determine the number of viable cells. Measurement of ATP by luciferase / luciferin based sensitive bioluminescent assays is known, see eg US Pat. No. 3,745,090. Luciferase (such as that from fireflies) is a microglobulin that uses ATP and molecular oxygen to catalyze the oxidative decarboxylation of luciferin to form oxyluciferin, a highly unstable single-stage An excited compound, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/00C12Q1/34
CPCC12Q1/008C12Q1/34G01N2333/914C12N9/14C12Y306/01C12Y306/01005G01N2333/90
Inventor P.阿萨拉普拉姆A.卢瑟姆
Owner APIRAYS BIOSCI AB
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