Protein expression and purification最新文献

{"title":"Codon harmonization for improve heterologous expression of human aromatase (CYP19A1) in E. coli cells","authors":"Marina I. Shaladonova,&nbsp;Yaraslau U. Dzichenka,&nbsp;Veronika V. Shchur,&nbsp;Antos B. Sachanka,&nbsp;Sergey A. Usanov","doi":"10.1016/j.pep.2025.106864","DOIUrl":"10.1016/j.pep.2025.106864","url":null,"abstract":"<div><div>CYP19A1 (aromatase) is the key enzyme taking part in the process of the conversion of androgens to estrogens thus considering as a major target for treatment of certain types of tumors, especially hormone dependent breast cancer. The purpose of this study is to achieve human aromatase (CYP19A1) from harmonized sequence and identify structural significance of rare codons, involved in the process of codon harmonization. There are several examples of influence of codon harmonization on the protein production and its activity, but these researches didn't include studying any human CYPs, which are difficult-to-produce membrane proteins. The aim of our study is to combine methods of codon harmonization, usage of optimal expression system and conditions, and modifications to the standard purification protocol in order to increase the yield of purified human recombinant aromatase for further usage of the enzyme for the screening of new inhibitors. In our research the structural significance of rare codons for the formation of the secondary structure is revealed, which is implemented in the algorithm of codon harmonization in human aromatase sequence. The combined approach with codon harmonization technique and one-step purification enables to achieve over 300 nmol of homogeneous, catalytically active human CYP19A1 per Liter of culture. We expect the results obtained will provide valuable information for protein engineering and enzymology.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106864"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145615544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression and production of recombinant SARS-CoV-2 RBD protein in E. coli system: comparison of performance, functionality and stability in three strains","authors":"Romina Golafshan ,&nbsp;Mahmoudreza Aghamali ,&nbsp;Mohammad Javad Rasaee","doi":"10.1016/j.pep.2025.106877","DOIUrl":"10.1016/j.pep.2025.106877","url":null,"abstract":"<div><div>The worldwide COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). The receptor binding domain (RBD) of SARS-Cov-2 is located on the spike protein, which binds and interacts with ACE2. The RBD serve as a crucial target for the development of virus-neutralizing antibodies, vaccinations, and inhibiting agents. The goal of this study was to synthesis recombinant RBD (rRBD) protein and evaluate its expression, function, and stability in different strains of <em>Escherichia coli</em> (<em>E. coli</em>). The rRBD domain gene sequence was engineered utilizing in silico methodologies. The optimized sequence was inserted into pET-28a vector using <em>Xho</em>I and <em>Eco</em>RI restriction sites. Three <em>E. coli</em> expression strains BL21 (DE3), Rosetta-gami, and Shuffle T7 were chosen for protein synthesis. Cloning of the target gene fragment was confirmed using PCR. The validated recombinant vector was cloned into three <em>E. coli</em> strains. SDS-PAGE and Western blot analysis were used to examine the expression of the rRBD. Far-UV circular dichroism (CD) spectroscopy was used to examine secondary structure. ELISA experiments quantified the interaction of rRBD protein with HRP-conjugated anti-His antibody and serum from COVID-19 patients detected with anti-human immunoglobulin-HRP labeled. Protein stability was assessed under different temperature conditions. The rRBD protein was best expressed in all three bacterial strains at 1 mM concentration of IPTG. A 14 kDa band corresponding to the rRBD protein was identified in all three bacterial strains via Western blot. BL21 had the greatest amount of rRBD expression. The rRBD generated from Rosetta-gami exhibited the most robust affinity for anti-His antibodies. All three expressed proteins exhibited similar secondary structure, characterized by a predominance of random coil and β-sheet content. They also exhibited similar affinity to antibodies against SARS-Cov-2 generated from patients. Storage at −20 °C and 4 °C with suitable glycerol concentrations ensured optimal long-term protein stability. This study reveals that <em>E. coli</em> is still a good and flexible host system to produce functional SARS-Cov-2 RBD protein. Even though the strains had different levels of expression and binding efficacy, all of the proteins they made had comparable structures and antigenic properties. The findings validate the feasibility of using bacterial expression systems for the cost-effective production of RBD-based diagnostic reagents.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106877"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145896848","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expression of recombinant preenzyme Blo t 1 allergen in Escherichia coli and its IgE reactivity","authors":"Chi Linh Thi Nguyen ,&nbsp;Uyen Quynh Nguyen ,&nbsp;Phuong Mai Vu ,&nbsp;Truong Huu Nguyen ,&nbsp;Phuong Hoang Nguyen ,&nbsp;Chi Quynh Thi Do ,&nbsp;Vinh Van Hoang","doi":"10.1016/j.pep.2025.106872","DOIUrl":"10.1016/j.pep.2025.106872","url":null,"abstract":"<div><div><em>Blomia tropicalis</em> is a predominant house dust mite in tropical-subtropical climates, and its allergen Blo t 1 is recognized as a key trigger of allergic responses. This study aimed to express, purify recombinant Blo t 1 from <em>B. tropicalis</em> collected in Vietnam and assess its IgE-binding capacity using Vietnamese allergic sera. The cDNA encoding Blo t 1 was amplified from local samples, cloned into pET32b (+), and expressed in <em>Escherichia coli</em> BL21 (DE3). IgE-binding properties were evaluated through dot blot and Western blot assays. The recombinant Blo t 1 showed anomalous SDS-PAGE migration at ∼63 kDa, comprising ∼38.11 kDa from 333 amino acids of the pre-enzyme form of Blo t 1 and ∼19.47 kDa derived from vector-associated sequences, including His-tag. The protein was successfully expressed and purified using His-tag affinity chromatography. Dot blot analysis showed that 19/30 sera (63.33 %) from <em>B. tropicalis</em>-sensitized patients reacted with the recombinant protein, whereas none of the 24 control sera did. Western blot confirmed IgE-binding specificity. The recombinant Blo t 1 was purified under denaturing conditions, so the native three-dimensional structure is unlikely to be retained, and the observed IgE reactivity mainly reflects linear epitope recognition. Even so, the protein represents a useful research material corresponding to a Blo t 1 variant circulating in Vietnam. Along with expanding the limited molecular and IgE-reactivity data available for <em>B. tropicalis</em> in Southeast Asia, it also provides a basis for studies on allergen structure, epitope features, and early exploratory work related to allergen-specific immunotherapy and SIT-oriented approaches.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106872"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145828358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The advantage of high pH eluting Protein A resins over their regular counterparts in aggregate and host cell protein clearance","authors":"Ziyang Li ,&nbsp;Lixia Hu ,&nbsp;Yifeng Li","doi":"10.1016/j.pep.2025.106859","DOIUrl":"10.1016/j.pep.2025.106859","url":null,"abstract":"<div><div>Protein A affinity chromatography is the most well-established method and gold standard for antibody purification. Typically, low pH (i.e., 3.0–3.5) is required for elution of bound antibodies from a Protein A column. However, for acid-sensitive antibodies, this low pH condition can trigger severe aggregation. Therefore, alternative Protein A resins supporting mild elution are highly desirable. To meet this need, recently Purolite and Cytiva both developed high pH eluting Protein A resins (i.e., Jetted A50 HipH and MabSelect mild elution, respectively). We previously showed that Jetted A50 HipH not only supports mild pH elution but also exhibits improved aggregate separation capability. In the current work, with four case studies, we compared the above-mentioned two high pH eluting Protein A resins with their regular counterparts (i.e., Jetted A50 and MabSelect SuRe LX, respectively) and demonstrated that the two special Protein A resins provided much better aggregate removal and host cell protein (HCP) clearance. Thus, in addition to supporting mild elution, the two high pH eluting Protein A resins also have an edge in terms of impurity clearance.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106859"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145557751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of a polyclonal antibody that specifically recognizes the recombinant NS5A protein of bovine viral diarrhea virus","authors":"Weijie Zhou,&nbsp;Yuting Wang,&nbsp;Shaobo Liang,&nbsp;Jingjin Hu,&nbsp;Feng Pang","doi":"10.1016/j.pep.2025.106868","DOIUrl":"10.1016/j.pep.2025.106868","url":null,"abstract":"<div><div>Bovine viral diarrhea virus (BVDV) is an economically important pathogen for causing bovine viral diarrhea (BVD), with a significant detrimental impact on cattle production worldwide. While the BVDV NS5A protein is known to be essential for replication, its function remain incompletely characterized. This study entailed cloning the NS5A gene and subsequently generating the recombinant plasmid pET-28a-NS5A for expression in <em>Escherichia coli</em>. The purified and refolded recombinant NS5A protein was employed to immunize New Zealand white rabbits, resulting in the production of specific polyclonal antibodies. The antibody was then deployed to investigate the expression of the NS5A protein. Our findings confirmed the NS5A protein was successfully produced and purified in a prokaryotic system. The generated anti-NS5A polyclonal antibody in rabbits showed a robust titer of 1:819,200. The antibody specifically detected the NS5A protein expressed in both prokaryotic and eukaryotic systems. With its high specificity and titer, this antibody may serve as a valuable reagent for investigating NS5A's functions in BVDV pathogenesis. It also holds potential for application in developing diagnostic assays and novel control strategies against BVD.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106868"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145725710","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A kaleidoscope of hosts: Expression systems of recombinant antibody reagents for immunological assays","authors":"Jia Xuan Yeoh ,&nbsp;Yee Siew Choong ,&nbsp;Theam Soon Lim","doi":"10.1016/j.pep.2025.106857","DOIUrl":"10.1016/j.pep.2025.106857","url":null,"abstract":"<div><div>Monoclonal antibodies (mAbs) have been shown to be highly promising reagents used in immunological analysis of various diseases and other chronic conditions due to their specific targeting, potency, and stability. Advances in protein engineering and immunology have led to the development of recombinant monoclonal antibodies at a staggering pace. Full-length antibodies IgG are generally the preferred format, but the development of smaller formats like Fab, scFv, and sdAbs opened the floodgates for more variation. This allowed for a more flexible application of mAbs in immunological assays. A diverse set of expression systems have been used to express recombinant mAbs which includes bacterial, yeast, insect, mammalian cells, plant, cell-free and <em>Leishmania</em> each with distinct advantages and disadvantages. This review highlights that the selection of an optimal expression system and antibody format must be guided by the intended application, balancing yield, structural integrity, and cost. While no single host fulfils all criteria, continued advances in host engineering, synthetic design, and AI-driven optimization are expected to streamline recombinant antibody production and expand its applicability across therapeutic and diagnostic fields.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106857"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145542195","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Amino acid chemistry and post-translational modifications underlying marine adhesive proteins: Biochemical insights for designing underwater adhesives","authors":"Taehee Yoon ,&nbsp;Hyung Joon Cha","doi":"10.1016/j.pep.2025.106851","DOIUrl":"10.1016/j.pep.2025.106851","url":null,"abstract":"<div><div>Underwater adhesion remains a complex challenge due to the interference of water with traditional bonding mechanisms. Marine organisms, such as mussels, sandcastle worms, and barnacles, have evolved specialized adhesive proteins that overcome these limitations through functional amino acid motifs. This review highlights how post-translationally derived amino acid motifs, such as 3,4-dihydroxylphenylalanine (DOPA) and phosphoserine, drive interfacial adhesion and the curing process through interfacial binding, metal coordination, and crosslinking. Alongside these, canonical amino acids, such as cysteine, lysine, arginine, histidine, glycine, and proline, contribute to redox buffering, electrostatic interactions, structural flexibility, and fibrillar assembly, which are essential for adhesive structure. The adhesive strategies of mussels, sandcastle worms, and barnacles reflect diverse mechanisms: mussels utilize DOPA-rich proteins with redox-regulation; sandcastle worms employ electrostatically driven coacervation and ion-mediated curing; barnacles generate nanostructured networks stabilized by disulfide bonding and hydrophobic packing. This review presents a unified molecular framework that links amino acid chemistry, biochemical transformations, and structural integration in underwater adhesion. It further discusses synthetic and recombinant approaches that mimic these natural systems, including catechol-functionalized polymers, complex coacervate adhesives, and genetically engineered proteins. These biomimetic platforms demonstrate the potential of translating marine adhesion logic into robust, water-compatible materials for biomedical and industrial applications.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106851"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145524120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strategic truncation improves recombinant expression of buffalo Fibulin-5 (FBLN5) in Escherichia coli","authors":"Deviyani Mahajan ,&nbsp;Neha Sarova ,&nbsp;Jyoti Rustagi ,&nbsp;Shwetali Prajapati ,&nbsp;Jai Kumar Kaushik ,&nbsp;Ashok Kumar Mohanty ,&nbsp;Manoj Kumar Jena ,&nbsp;Sudarshan Kumar","doi":"10.1016/j.pep.2025.106858","DOIUrl":"10.1016/j.pep.2025.106858","url":null,"abstract":"<div><h3>Background</h3><div>Accurate early pregnancy detection is crucial for enhancing herd productivity in bovines. There is growing interest in identifying reliable protein biomarkers that can be used to develop rapid, sensitive, and cost-effective diagnostic tools. This study focused on buffalo fibulin-5 (FBLN5), an extracellular matrix glycoprotein involved in tissue remodeling and cell adhesion, and a potential early pregnancy biomarker.</div></div><div><h3>Methods</h3><div>In this study, we used the pET-32a(+) expression vector and three <em>E. coli</em> expression hosts. Heat-shock transformation was performed in chemically competent cells (CaCl₂ method), and the expression was optimized at 18 °C and 37 °C. Recombinant truncated FBLN5 was purified through immobilized metal affinity chromatography (IMAC) on nickel resin and subsequently characterized by SDS-PAGE and Western blotting.</div></div><div><h3>Results</h3><div>Optimization of various expression conditions, such as temperature, induction, incubation time, and additives, did not result in any detectable expression of full-length FBLN5. Therefore, a truncated version of FBLN5 was designed based on the predicted antigenic regions to increase its immunogenicity and simplify its recombinant expression. Truncated FBLN5 (<em>Trx</em>-tagged and 6 × His-tagged) expressed well in BL21Codon Plus (DE3)-RIL and Lemo21(DE3) at 18 °C with 1 mM IPTG induction. Furthermore, we successfully purified soluble truncated FBLN5 and confirmed its molecular weight (∼36 kDa) and identity using western blotting with enhanced chemiluminescence (ECL).</div></div><div><h3>Conclusion</h3><div>Truncation of full-length FBLN5 resulted in successful cloning, expression, and purification of this otherwise difficult to express protein. This study provides a solid foundation for future immunological studies aimed to evaluate FBLN5 potential as diagnostic marker in early pregnancy detection in bovine species.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106858"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145557658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation, purification and identification of major allergens Cor a 9 and Cor a 14 in hazelnuts","authors":"Weichao Zhu ,&nbsp;Huahong Yu ,&nbsp;Ying Zhang ,&nbsp;Qishu Luo ,&nbsp;Min Song ,&nbsp;Qin Geng ,&nbsp;Lihua Zhou ,&nbsp;Zhihua Wu","doi":"10.1016/j.pep.2025.106866","DOIUrl":"10.1016/j.pep.2025.106866","url":null,"abstract":"<div><div>Hazelnut (<em>Corylus avellana</em>) is a widely consumed nut with potent allergenic potential, and the isolation and purification of native hazelnut allergens are critical for the effective prevention and management of hazelnut allergy. In this study, two major allergens Cor a 9 and Cor a 14 were purified from hazelnut kernels via a sequential protocol involving pulverization, defatting, protein extraction, ammonium sulfate precipitation, dialysis desalting, and anion-exchange chromatography. Results demonstrated successful purification of the target allergens with high purity and intact conformational structure, which exhibited specific immunoreactivity with sera from hazelnut-allergic patients. A single preparation run yielded over 5 mg of Cor a 14 (purity &gt;95 %) and more than 1 mg of Cor a 9 (purity &gt;85 %). The established protocol is simple, requires minimal equipment, and offers high efficiency, laying a preliminary foundation for hazelnut allergen-related research. In conclusion, we successfully isolated and purified two major hazelnut allergenic proteins Cor a 9 and Cor a 14, providing a valuable material basis for investigating the sensitization mechanism of hazelnut allergy and developing diagnostic tools or hypoallergenic products.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106866"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145681148","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bench-top NMR of water signals: A non-destructive tool for biomacromolecule characterization","authors":"Tomoto Ura ,&nbsp;Taiji Oyama ,&nbsp;Chiaki Nishimura","doi":"10.1016/j.pep.2025.106855","DOIUrl":"10.1016/j.pep.2025.106855","url":null,"abstract":"<div><div>Bench-top NMR spectroscopy has emerged as an accessible, non-destructive tool for analyzing biomacromolecules in solution. This review focuses on how water proton NMR (wNMR) provides sensitive readouts of protein states in solution. We highlight representative studies on monoclonal antibodies, insulin, and vaccines under diverse stresses, where wNMR measurements consistently correlate with aggregation and stability outcomes. In parallel, we revisit the fundamental principles of wNMR, focusing on the molecular origins of water relaxation and the roles of hydration dynamics, proton exchange, and water confinement near protein surfaces. To illustrate these mechanisms, we present simple illustrative experiments using bench-top wNMR and ATR-FTIR spectroscopy with aqueous salts and polymers, revealing how non-protein solutes modulate hydrogen-bond networks and water dynamics. Finally, we discuss emerging perspectives that extend the scope of bench-top NMR beyond protein formulations. Together, these developments position bench-top NMR not only as a practical tool for stability assessment but also as a promising framework for probing water-mediated phenomena across biopharmaceutical and soft-matter systems.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"239 ","pages":"Article 106855"},"PeriodicalIF":1.2,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145565166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}