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

Abstract 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.

[1]  T. Pisitkun,et al.  Expression of Secreted Neutrophil Gelatinase-Associated Lipocalin in 293T Cell Using the Inducible Dual-Function System , 2021 .

[2]  V. Chernov,et al.  Production and Application of Multicistronic Constructs for Various Human Disease Therapies , 2019, Pharmaceutics.

[3]  Sooyoung Chung,et al.  Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence , 2019, Molecules and cells.

[4]  L. Qi,et al.  Urine NGAL as an early biomarker for diabetic kidney disease: accumulated evidence from observational studies , 2019, Renal failure.

[5]  M. Pirmohamed,et al.  Urinary Biomarkers of Aminoglycoside-Induced Nephrotoxicity in Cystic Fibrosis: Kidney Injury Molecule-1 and Neutrophil Gelatinase-Associated Lipocalin , 2018, Scientific Reports.

[6]  A. Rahimpour,et al.  Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells , 2018, Preparative biochemistry & biotechnology.

[7]  C. Faes,et al.  Establishment of reference values for novel urinary biomarkers for renal damage in the healthy population: are age and gender an issue? , 2013, Clinical chemistry and laboratory medicine.

[8]  Xiaole Kong,et al.  Chemistry and biology of siderophores. , 2010, Natural product reports.

[9]  A. Skerra,et al.  Neutrophil Gelatinase-Associated Lipocalin Expresses Antimicrobial Activity by Interfering with l-Norepinephrine-Mediated Bacterial Iron Acquisition , 2010, Antimicrobial Agents and Chemotherapy.

[10]  M. Legrand.  Faculty Opinions recommendation of Plasma and urine neutrophil gelatinase-associated lipocalin in septic versus non-septic acute kidney injury in critical illness. , 2010 .

[11]  D. Bolignano,et al.  Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage. , 2008, American journal of kidney diseases : the official journal of the National Kidney Foundation.

[12]  J. Cowland,et al.  Neutrophil gelatinase-associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9. , 2007, Arthritis and rheumatism.

[13]  M. Mitsnefes,et al.  NGAL is an early predictive biomarker of contrast-induced nephropathy in children , 2007, Pediatric Nephrology.

[14]  M. Marahiel,et al.  Siderophore-Based Iron Acquisition and Pathogen Control , 2007, Microbiology and Molecular Biology Reviews.

[15]  K. Mori,et al.  Dual action of neutrophil gelatinase-associated lipocalin. , 2007, Journal of the American Society of Nephrology : JASN.

[16]  David R. Liu,et al.  The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2 , 2006, Proceedings of the National Academy of Sciences.

[17]  D. Wood,et al.  Inteins and affinity resin substitutes for protein purification and scale up , 2005, Microbial cell factories.

[18]  Qing Ma,et al.  Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery , 2005, The Lancet.

[19]  Shizuo Akira,et al.  Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron , 2004, Nature.

[20]  M. Mitsnefes,et al.  Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. , 2003, Journal of the American Society of Nephrology : JASN.

[21]  X. Zhan,et al.  Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[22]  W. Bentley,et al.  Biotechnological applications of green fluorescent protein , 2003, Applied Microbiology and Biotechnology.

[23]  R. Strong,et al.  The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. , 2002, Molecular cell.

[24]  Teruyuki Nagamune,et al.  Design of the linkers which effectively separate domains of a bifunctional fusion protein. , 2001, Protein engineering.

[25]  N. Dharmasiri,et al.  Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures. , 2001, Journal of biotechnology.

[26]  A. Maule,et al.  Limitations on the use of fused green fluorescent protein to investigate structure-function relationships for the cauliflower mosaic virus movement protein. , 2000, The Journal of general virology.

[27]  H. Mizuguchi,et al.  IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. , 2000, Molecular therapy : the journal of the American Society of Gene Therapy.

[28]  W. Bentley,et al.  Insect larval expression process is optimized by generating fusions with green fluorescent protein. , 1999, Biotechnology and bioengineering.

[29]  E. Martínez-Salas.  Internal ribosome entry site biology and its use in expression vectors. , 1999, Current opinion in biotechnology.

[30]  I. Pastan,et al.  Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector. , 1998, Human gene therapy.

[31]  E. Veerman,et al.  EP-GP and the Lipocalin VEGh, Two Different Human Salivary 20-kDa Proteins , 1995, Journal of dental research.

[32]  L. Kjeldsen.  Gelatinase granules in human neutrophils , 1995, European journal of haematology. Supplementum.

[33]  C. Albano,et al.  Green Fluorescent Protein as a Real Time Quantitative Reporter of Heterologous Protein Production , 1998, Biotechnology progress.