Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Protein A-Nanoluciferase fusion protein for generalized, sensitive detection of immunoglobulin G.

Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).

Isolation and Barcoding of Trace Pollen-free DNA for Authentication of Honey.

Adulteration and mislabeling of honey to mask its true origin have become a global concern. Pollen microscopy, the current gold standard for identifying honey's geographical and plant origins, is laborious, requires extensive training, and fails to identify filtered honey and honey spiked with pollen from a more favorable plant to disguise its origins. We successfully isolated pollen-free DNA from filtered honey using three types of adsorbents: (i) anti-dsDNA antibodies coupled to magnetic microspheres; (ii) anion-exchange adsorbent; and (iii) ceramic hydroxyapatite. The internal transcribed spacer 2 region of the captured pollen-free DNA was polymerase chain reaction-amplified and subjected to next-generation sequencing. Using an in-house bioinformatics pipeline, initial experiments showed that anion exchange had the greatest capacity to capture trace pollen-free DNA, and it was successfully applied to isolate DNA from five honey samples. Enrichment of trace pollen-free DNA from filtered honey samples opens a new approach for identifying the true origins of honey.

Sediment and their bacterial communities in an industrialized estuary after Hurricane Harvey.

Estuaries experience variable physicochemical conditions, especially after hurricanes and due to anthropogenic sources of pollution. Their microbial communities are not as well understood in terms of community structure and diversity, particularly in response to stresses from pollution and severe events. This study presents a 16S rRNA-based description of sediment microbial communities in the Houston Ship Channel-Galveston Bay estuary after Hurricane Harvey in 2017. A total of 11 sites were sampled, and microbial genomic DNA was isolated from sediment. The presence and abundance of specific bacterial and archaeal taxa in the sediment indicated pollutant inputs from identified legacy sources. The abundance of certain microbial groups was explained by the mobilization of contaminated sediment and sediment transport due to Harvey. Several microorganisms involved in the biodegradation of xenobiotics were observed. The spatial occurrence of Dehalococcoidia, a degrader of persistent polychlorinated compounds, was explained in relation to sediment properties and contaminant concentrations.

Longitudinal patterns in sediment type and quality during daily flow regimes and following natural hazards in an urban estuary: a Hurricane Harvey retrospective.

Understanding the transport of sediments in urban estuaries and their effects on water quality and microorganisms is a convergent challenge that has yet to be addressed especially as a result of natural hazards that affect the hydrodynamics of estuarine systems. This study provides a holistic view of the longitudinal nature and character of sediment in an urban estuary, the Galveston Bay Estuary System (GBES), under daily and extreme flow regimes and presents the results of water and sediment sampling after Hurricane Harvey. The sediment sampling quantified total suspended sediment (TSS) concentrations, metal concentrations, and the diversity of microbial communities. The results revealed the impact of the substantial sediment loads that were transported into the GBES in terms of sediment grain type, the spatial distribution of trace metals, and the diversity of microbial communities. A measurable shift in the percentage of silt relative to historical norms was noted in the GBES after Hurricane Harvey. Not only did sediment metal data confirms this shift and its ensuing impact on metal concentrations; microbial data provided ample evidence of the effect of leaks and spills from wastewater treatment plants, superfund sites, and industrial runoff on microbial diversity. The research demonstrates the importance of understanding longitudinal sediment transport and deposition in estuarine systems under daily flow regimes but more critically, following natural hazard events to ensure sustainability and resilience of systems such as the GBES that encounter numerous acute and chronic stresses.

Continuous Fc detection for protein A capture process control.

Purification of therapeutic monoclonal antibodies usually involves a protein A affinity capture step. Because column breakthrough of antibody in complex, UV-absorbing culture fluid cannot be readily detected in real time, processes are designed so conservatively that column capacity is usually underutilized, wasting adsorbent and reducing productivity. We have developed a fluorescence-based monitoring technology which allows real-time mAb monitoring and used it to detect IgG in column breakthrough. The column effluent was continuously contacted with soluble fluorescein-labeled Fc-binding ligands to produce an immediately-detectable shift in both fluorescence polarization and intensity. To extend the upper limit of inlet flow rate, a 14:1 split-ratio flow splitter was tested with an inlet flow of 15 mL/min (0.9 L/h), producing a sampling stream at 1 mL/min while still enabling continuous detection functionality. We observed significant shifts in fluorescence intensity in CHO cell culture fluid spiked with human IgG, and detected 0.02-0.1 g/L human IgG in protein A column breakthrough at a flow velocity of 80 cm/h. The increase in fluorescence intensity upon 0.1% breakthrough of an 1 g/L feed was used to trigger column switching using Python-enabled two-way communication with the standard Unicorn OPC process control protocol. The technology allows rapid, continuous and reliable monitoring of IgG in a flowing process stream, without elaborate sample preparation.

Neutral DNA-avidin nanoparticles as ultrasensitive reporters in immuno-PCR.

We have developed an immuno-PCR based diagnostic platform which couples detection antibodies to self-assembled, ultra-detectable DNA-avidin nanoparticles stabilized with poly(ethylene glycol) to link DNA amplification to target protein concentration. Electrostatic neutralization and cloaking of the PCR-amplifiable DNA labels by avidin and PEG coating reduces non-specific "stickiness" and enhances assay sensitivity. We further optimized the detectability of the nanoparticles by incorporating four repeats of a unique synthetic DNA PCR target into each nanoparticle. Using human chorionic gonadotropin hormone (hCG) as a model analyte, this platform was able to quantitate the target hCG protein in femtomolar concentrations using only standard laboratory equipment.

Recombinant expression, characterization, and quantification in human cancer cell lines of the Anaplastic Large-Cell Lymphoma-characteristic NPM-ALK fusion protein.

Systemic anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma most commonly seen in children and young adults. The majority of pediatric ALCLs are associated with the t(2;5)(p23;q35) translocation which fuses the Anaplastic Lymphoma Kinase (ALK) gene with the Nucleophosmin (NPM) gene. The NPM-ALK fusion protein is a constitutively-active tyrosine kinase, and plays a major role in tumor pathogenesis. In an effort to advance novel diagnostic approaches and the understanding of the function of this fusion protein in cancer cells, we expressed in E. coli, purified and characterized human NPM-ALK fusion protein to be used as a standard for estimating expression levels in cultured human ALCL cells, a key tool in ALCL pathobiology research. We estimated that NPM-ALK fusion protein is expressed at substantial levels in both Karpas 299 and SU-DHL-1 cells (ca. 4-6 million molecules or 0.5-0.7 pg protein per cell; based on our in-house developed NPM-ALK ELISA; LOD of 40 pM) as compared to the ubiquitous ß-actin protein (ca. 64 million molecules or 4.5 pg per lymphocyte). We also compared NPM-ALK/ ß-actin ratios determined by ELISA to those independently determined by two-dimensional electrophoresis and showed that the two methods are in good agreement.

Corrigendum: Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.

[This corrects the article DOI: 10.3389/fcimb.2018.00237.].

Nanoparticle-Based Proximity Ligation Assay for Ultrasensitive, Quantitative Detection of Protein Biomarkers.

Proximity ligation assay (PLA) achieves extremely low limits of detection but requires the synthesis of antibody-DNA conjugates recognizing multiple target epitopes with appropriate proximity. In this work, we introduce a more generally applicable method by replacing antibody-DNA conjugates with nanoparticles which create ultradetectable PCR templates by capturing biotinylated oligonucleotides and catalyzing ligation. A competitive PCR readout was used to make the assay quantitative. We have demonstrated that NP-PLA can detect and quantitate human chorionic gonadotropin (hCG) levels as low as 2.6 fM (∼0.1 pg/mL), nearly 1000 times more sensitive than the current state of the art ELISA.

Akkermansia muciniphila as a Model Case for the Development of an Improved Quantitative RPA Microbiome Assay.

Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation and storage that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried enzyme pellets that can be used to analyze specimens rapidly in the field or clinic, using a portable fluorometer. Immediate analysis of diverse bacterial communities can lead to a more accurate quantification of relative bacterial abundance. In this study, we discovered that universal bacterial 16S ribosomal DNA primers give false-positive signals in RPA analysis because manufacturing host Escherichia coli DNA is present in the RPA reagents. The manufacturer of RPA reagents advises against developing an RPA assay that detects the presence of E. coli due to the presence of contaminating E. coli DNA in the reaction buffer (www.twistdx.co.uk/). We, therefore, explored four strategies to deplete or fragment extraneous DNA in RPA reagents while preserving enzyme activity: metal-chelate affinity chromatography, sonication, DNA cleavage using methylation-dependent restriction endonucleases, and DNA depletion using anti-DNA antibodies. Removing DNA with anti-DNA antibodies enabled the development of a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium Akkermansia muciniphila in human feces.

Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity.

Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme's thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its k cat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein-protein interfaces leading to the protein quaternary structure.

Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein.

The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195-206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.

Evaluation of the LIAISON chemiluminescence immunoassay for diagnosis of syphilis.

We report the results of an evaluation of the LIAISON Treponema pallidum-specific assay, a one-step sandwich chemiluminescence immunoassay (CLIA), as a screening test and as a confirmatory test for the diagnosis of syphilis. The assay was compared with the CAPTIA Syphilis-G enzyme immunoassay (EIA) and with a testing algorithm that also included the rapid plasma reagin (RPR) and T. pallidum particle agglutination (PA) assays. As a screening test, the CLIA showed levels of agreement with the EIA and with the algorithm, respectively, of 94.1 and 100% for 51 samples from patients with primary or secondary syphilis, 93.2 and 98.7% for 999 samples sent to the laboratory for routine syphilis testing, 84.5 and 94.0% for 200 samples from human immunodeficiency virus-positive patients, 98.0 and 100% for 200 samples from pregnant patients, and 94.3 and 98.3% for 992 samples from apparently healthy adults. As a confirmatory test, the CLIA showed 99% agreement with the EIA for 204 RPR-positive samples. After resolution with further T. pallidum PA testing and the discarding of one sample of insufficient quantity, there was 100% agreement for the remaining 203 samples. For the total group of 2,645 samples, the overall relative sensitivity was 95.8% and the relative specificity was 99.1%. We conclude that the LIAISON CLIA demonstrated excellent sensitivity and specificity when evaluated as a confirmatory test and as a screening test for syphilis among various patient populations, including specific populations with reportedly increased rates of false-positive nontreponemal test results.