Bicistronic vector-based procedure to measure correlative expression and bacteriostatic activity of recombinant neutrophil gelatinase-associated lipocalin

Neutrophil gelatinase-associated lipocalin (NGAL) is a component of the human innate immune system that exerts bacteriostatic effects. Recent research suggests that NGAL is also a promising biomarker for tubular damage and aids in the early diagnosis of acute kidney injury. Therefore, the developmen...

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Published inBiotechnology, biotechnological equipment Vol. 36; no. 1; pp. 545 - 552
Main Authors Saoin, Somphot, Arunyanak, Naphatswan, Muangchan, Pornuma, Boonkrai, Chatikorn, Pisitkun, Trairak, Kloypan, Chiraphat, Nangola, Sawitree
Format Journal Article
LanguageEnglish
Published Sofia Taylor & Francis 31.12.2022
Taylor & Francis Ltd
Taylor & Francis Group
Subjects
bacteriostatic effect
Bacteriostats
bicistronic vector
Biological activity
Biomarkers
Enzyme-linked immunosorbent assay
Fluorescence
fluorescence intensity
Gelatinase
Green fluorescent protein
Immune system
Innate immunity
Iron
Leukocytes (neutrophilic)
Lipocalin
Neutrophil gelatinase-associated lipocalin
Neutrophils
Proteins
siderophore
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Summary:Neutrophil gelatinase-associated lipocalin (NGAL) is a component of the human innate immune system that exerts bacteriostatic effects. Recent research suggests that NGAL is also a promising biomarker for tubular damage and aids in the early diagnosis of acute kidney injury. Therefore, the development of a method to efficiently produce recombinant NGAL has considerable clinical value. This study aimed to analyze the correlation between fluorescence reporter (enhanced green fluorescent protein, EGFP) intensity and NGAL levels, mediated by a bicistronic expression system based on internal ribosome entry sites (IRESs). During the 14-day culture period, EGFP intensity was positively correlated with NGAL concentrations. The purified protein also exerted bacteriostatic effects, interfering with bacterial iron uptake machinery during periods of iron starvation. The purified NGAL was also applied to generate a standard curve that could be used for enzyme-linked immunosorbent assay of samples containing unknown NGAL quantities. In conclusion, we successfully developed a procedure using EGFP and IRES-based vector to predict NGAL production levels and optimize protein harvest time. Our approach is also a simple and effective method for evaluating bioactivity.
ISSN:1310-2818
1314-3530
DOI:10.1080/13102818.2022.2101943