We have developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.
Recombinant protein expression; Protein purification; DNA polymerase η; DNA polymerase ι; DNA polymerase ν
To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.
Biacore; Kinetics; Optical biosensor; Surface plasmon resonance
A quantitative bioluminescence assay for rapid and sensitive microRNA (miRNA) expression analysis was developed. The assay utilizes miRNA directly as a primer for binding to a circular single-stranded DNA template, followed by rolling circle amplification. The detection of inorganic pyrophosphate (PPi) molecules released during the DNA polymerization and amplification process is performed by a multi-enzyme system. PPi is converted to ATP by ATP-sulfurylase, which provides energy for luciferase to oxidize luciferin and produce light. Experimental results show that the assay has a dynamic range exceeding three orders of magnitude and the ability to discriminate miRNAs with high-homology sequences. Quantification of nine miRNAs in human heart tissues demonstrated a high cross-platform consistency between this assay and the TaqMan real time PCR assay with R2 = 0.941. The assay requires fewer reagents, can be performed at an isothermal condition without thermal cycling, and is capable of detecting miRNAs in less than an hour. Compared to the real time PCR and microarray-based detection methods, this assay provides a simpler, faster, and less expensive platform for miRNA quantification in life science research, drug discovery, and clinical diagnosis.
Bioluminescence assay; microRNA; Quantification; Rolling circle amplification
Thrombelastography (TEG) is a method that is used to conduct global assays which monitor fibrin formation and fibrinolysis and platelet aggregation in whole blood. The purpose of this study was to use a well-characterized tissue factor (Tf) reagent and contact pathway inhibitor (corn trypsin inhibitor, CTI) to develop a reproducible thrombelastography assay. In this study, blood was collected from 5 male subjects (3 times). Clot formation was initiated in whole blood with 5 pM Tf in the presence of CTI and fibrinolysis was induced by adding tissue-plasminogen activator (tPA). Changes in visco-elasticity were then monitored by TEG. In quality control assays, our Tf reagent, when used at 5 pM, induced coagulation in whole blood in 3.93±0.23 minutes and in plasma in 5.12±0.23 minutes (n=3). In TEG assays, tPA significantly decreased clot strength (maximum amplitude, MA) in all individuals but had no effect on clot time (R time). The intra-assay variability (CVa<10%) for R time, angle and MA suggests that these parameters reliably describe the dynamics of fibrin formation and degradation in whole blood. Our Tf reagent reproducibly induces coagulation, making it an ideal tool to quantify the processes that contribute to mechanical clot strength in whole blood.
Thrombelastography; fibrin; PAI-1; fibrinolysis; tPA
Poly(ADP-ribose) [pADPr] is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD+ by poly(ADP-ribose) polymerases [PARPs] and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. PADPr is involved in many cellular processes and exerts biological function through covalent modification and non-covalent binding to specific proteins. Very little is known about molecular recognition and structure activity relationships for non-covalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.
PARP; PARG; pADPr; PAR; ADPr; poly(adp-ribose); poly(ADP-ribose) polymerase; poly(ADP-ribose) glycohydrolase; polymer; NAD+; ADP ribose
Fatty Acid Synthase (FASN, FAS; EC 18.104.22.168) is the sole mammalian enzyme to synthesize fatty acids de novo from acetyl and malonyl coenzyme A esters. A new method is described that directly quantifies uniformly labeled [13C]16-palmitate by tracing [13C]2-acetyl-CoA and [13C]3-malonyl-CoA using an in vitro FASN assay. This method used GC-MS to detect [13C]16-palmitate carboxylate anions (m/z 271) of pentafluorobenzyl derivatives and was highly sensitive at femtomole quantities. Uniformly incorporated [13C]16-palmitate was the primary product of both recombinant and crude tissue lysate FASN. Quantification of FASN protein within crude tissue lysates assured equal FASN amounts, preserved steady state kinetics, and enabled calculation of FASN specific activity. FASN activity determined by [13C]16-palmitate synthesis was consistent with values obtained from NADPH oxidation assays. Analysis of FASN activity from tissue extracts was not hampered by contaminating enzymes or pre-existing fatty acids. Crude mammary gland and liver lysates had significantly different activities at 82 and 65 nmoles minute−1 mg−1 respectively, suggesting tissue specific activity levels differ in a manner unrelated to FASN amount. GC-MS quantification of [13C]16-palmitate synthesis permits sensitive evaluation of FASN activity from tissues of varied physiologic states, and of purified FASN activity in the presence of modifying proteins, enzymes, or drugs.
Fatty Acid Synthase Activity; GC-Mass Spectrometry; Mammary Gland/Liver Lysates; [13C]-substrate incorporation; [13C]16-Palmitic Acid
We have devised protein-folding sensors that link protein stability to TEM-1 β-lactamase activity. Addition of osmolytes and other compounds with chemical chaperone activity to the growth media of bacteria containing these sensors increases β-lactamase activity up to 207-fold in a dose-dependent manner. This enables the rapid detection and sensitive quantification of compounds that enhance in vivo protein stability.
in vivo; folding sensor; β-lactamase; osmolytes; protein stability
Necrotic cell death is prevalent in many different pathologic disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways with the key regulator being RIP1, a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust HTS assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein-derivatives of Necrostatin-1 (Nec-1) and Necrostatin-3 (Nec-3). These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS since the assays have a DMSO tolerance up to 5% and Z' scores of 0.62 (fluorescein-Nec-1) and 0.57 (fluorescein-Nec-3). Additionally, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4.
Necrostatins (Necs); Receptor Interaction Protein 1 (RIP1) kinase; fluorescence polarization (FP); competition assays; high-throughput screening (HTS); ligand docking
Synthesis of (GT)5 tailed duplex DNA promoter is an important first step for purifying transcription complexes by promoter trapping purification. In our earlier publication we have shown that the purification of the c-jun promoter using lambda exonuclease digestion of PCR produced DNA with single stranded tails. Asymmetric polymerase chain reaction (PCR) can also produce tailed single strands which can be annealed to yield the desired promoter. An effective method utilizes asymmetric PCR and double digestion. After PCR, first a restriction enzyme, in this case SacII, cuts duplex strands remaining after asymmetric PCR, leaving 5’-phosphoryl ends susceptible to a second digestion with lambda exonuclease to effectively degrade any duplex. The resulting single strands are then annealed to produce a duplex DNA with a single stranded (GT)5 tail at the 3’ end of each strand of the duplex. Unlike the previously described method, this novel procedure produces the desired tailed promoter devoid of any untailed duplex.
transcription; transcription factors; Southwestern blotting; affinity chromatography
Histone octamers are the basic building blocks of chromatin and platforms for diverse genetic mechanisms. We report a simple method for preparing recombinant histone octamers by overexpressing all four histones from a single polycistronic vector followed by standard chromatography under native conditions. This approach reduces the time needed for the octamer preparation to a single day and should be applicable to making a variety of unmodified and modified histone octamers.
Histones; chromatin; nucleosome; polycistronic vector
Common de-N-glycosylation protocols usually require a lengthy incubation time. Although pressure cycling-technology or scientific microwave reactors can accelerate this enzyme reaction, they may not be easily accessible. In this brief report, we employed an alternative strategy using a standard domestic microwave oven to perform the de-N-glycosylation. Model glycoproteins (bovine RNase B, bovine fetuin, and human IgG) and a complex mixture from human plasma were fully deglycosylated in 20 min, without any apparent adverse affects on the glycans or protein backbones. This new method provides a simple and inexpensive solution to achieve rapid de-N-glycosylation.
De-N-glycosylation; domestic microwave; PNGase F; glycoprotein; N-glycan
Aristolochic acids (AAs), major components of plant extracts from Aristolochia species form, after metabolic activation, pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present herein a fast, non-isotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition, analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.
DNA adducts; aristolochic acids; fluorescence detection
The maximum entropy method (MEM) has been used in many studies to reliably recover effective lifetimes from kinetics, whether measured experimentally or simulated computationally. Here, recent claims made by Mulligan et al. regarding MEM analyses of kinetics (Anal. Biochem. 421 (2012) 181–190) are shown to be unfounded. Their assertion that their software allows “analysis of datasets too noisy to process by existing iterative search algorithms” is refuted with a MEM analysis of their triexponential test case with increased noise. In addition, it is shown that lifetime distributions recovered from noisy kinetics data with the MEM can be improved by using a simple filter when bootstrapping the prior model. When deriving the bootstrapped model from the lifetime distribution obtained using a uniform model, only the slower processes are represented as Gaussians in the bootstrapped model. Using this new approach, results are clearly superior to those of Mulligan et al. despite the presence of increased noise. In a second example, ambiguity in the interpretation of Poisson kinetics in the presence of scattered excitation light is resolved by filtering the prior model.
Kinetics; Inverse Laplace transform; Rate constants; Lifetime distributions; Maximum entropy method
A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides is presented that relies on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. This method enables complete separation of seventeen GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone (AMAC) to improve sensitivity. The limit of detection was at the attomole level and about 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The RSD of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7% and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps, and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate, resulting from the separate analyses of a single sample.
2-aminoacridone; capillary electrophoresis; chondroitin /dermatan sulfate; heparan sulfate /heparin; hyaluronan; glycosaminoglycan
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) uses threshold cycles (Ct values) for measuring relative gene expression. Ct values are signal-to-noise data composed of target gene expression and multiple sources of confounding variations. Data analysis is to minimize technical noises, evaluate biological variances, and estimate treatment-attributable expression changes of particular genes. However, this function is not sufficiently fulfilled in current analytic methods. An important but unrecognizable problem is that Ct values from all biological replicates and technical repeats are pooled across genes and treatment types. This violates the sample-specific association between target and reference genes, leading to inefficient removal of technical noises. To resolve this problem, here we propose to separate Ct values into replicate-specific data subsets and iteratively analyze expression ratios for individual data subsets. The individual expression ratios, rather than the raw Ct values, are pooled to determine the final expression change. The variances of all biological replicates and technical repeats across all target and reference genes are summed up. Our results from example data demonstrate that this separated method can substantially minimize RT-qPCR variance compared with the traditional methods using pooled Ct profiles. This analytic strategy is more effective in control of technical noises and improves the fidelity of RT-qPCR quantification.
Real-time PCR; Data analysis; Normalization; Variance; Reverse transcription; Replicates; Gene expression; mRNA; cDNA; RT-qPCR
iTRAQ (isotope tags for relative and absolute quantification) reagent coupled with MALDI TOF/TOF mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies, and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, Lantus®) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including Human insulin (P28K29) and Lispro (K28P29), which only differ by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting.
iTRAQ/MALDI; insulins; Glu-C; deamidation; aggregation; generic protein drugs; counterfeit
The human placenta is a complex organ whose proper function is crucial for the development of the fetus. The placenta contains within its structure elements of the maternal and fetal circulatory systems. The interface with maternal blood is the lining of the placenta, that is a unique compartment known as the syncytiotrophoblast. This large syncytial structure is a single cell layer in thickness, and the apical plasma membrane of the syncytiotrophoblast interacts directly with maternal blood. Relatively little is known about the proteins that reside in this unique plasma membrane or how they may change in various placental diseases. Our goal was to develop methods for isolating highly enriched preparations of this apical plasma membrane compatible with high quality proteomics analysis and herein describe the properties of these isolated membranes.
Placenta; plasma membrane; syncytiotrophoblast
Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIIItubulin represented 3.2% of the total HeLa β-tubulin.
Label-free quantification; Mass spectrometry; Targeted proteomic analysis; Tubulin isotype quantification; Tubulin
Bottom-up proteomics requires the digestion of proteins into peptides by processes that use salts for denaturing and buffering purposes. These salts need to be removed prior to mass spectrometry analysis to reduce ion-suppression; solid phase extraction (SPE) is a commonly used strategy. There are many commercially-available SPE sorbent types and sizes, which are generally provided with manufacturer recommendations for use, including protein loading capacity. We found that these general suggestions were often not ideal, and our data suggest that context-specific evaluation of sorbent type and amount can improve reproducibility. Specifically, the universal Oasis HLB sorbent provided better retention of the more hydrophilic peptides than the traditional C18 reversed-phase SPE, but it did so at the expense of an increased loss of the more hydrophobic peptides. We found that increasing the amount of the C18 sorbent beyond the manufacturer's guidelines decreased breakthrough (i.e., increased retention) of twelve hydrophilic, identifiable peptides without loss of hydrophobic peptides. This procedure was robust in a 96-well plate format.
Blocking efficacy of whole soymilk, non-fat soymilk, SuperBlock™, and non-fat milk was evaluated by performing standard protein immunoblotting procedures on both purified protein and crude nuclear extracts from HEK 293 cells. Non-fat soymilk was found to have superior blocking efficacy compared to other blocking agents in terms of high signal to noise ratio with the shortest blocking times. In addition, the presence of low concentrations of the detergent Tween-20 (0.05–0.1% v/v) in the wash buffer, as well as, antibody incubations significantly lessened the background compared to only including the detergent during wash steps.
immunoblots; western blots
The systemic amyloidoses are a rare, but deadly class of protein folding disorders with significant unmet diagnostic and therapeutic needs. The current model for symptomatic amyloid progression includes a causative role for soluble toxic aggregates as well as for the fibrillar tissue deposits. Although much research is focused on elucidating the potential mechanism of aggregate toxicity, evidence to support their existence in vivo has been limited. We report the use of a technique we have termed Biological On-Line Tracer Sedimentation (BOLTS) to detect abnormal high molecular weight complexes (HMWCs) in serum samples from individuals with systemic amyloidosis due to aggregation and deposition of wild-type transthyretin (senile systemic amyloidosis, SSA) or monoclonal immunoglobulin light chain (AL amyloidosis). In this proof-of-concept study, HMWCs were observed in 31 out of 77 amyloid samples (40.3%). HMWCs were not detected in any of the 17 non-amyloid control samples subjected to BOLTS analyses. These findings support the existence of potentially toxic amyloid aggregates, and suggest that BOLTS may be a useful analytic and diagnostic platform in the study of the amyloidoses or other diseases where abnormal molecular complexes are formed in serum.
Analytical ultracentrifugation; Amyloidosis; Serum aggregates; Sedimentation; Transthyretin
As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high quality DNA extraction from archival FFPE tissue specimen remains complex and time consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit; O.D 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with MSP can be used for epigenetic studies with the advantages of rapidity and high quality, and may contribute to the development of biomarkers in clinical studies.
DNA extraction; DNA methylation; Methylation Sepcific PCR (MSP); Formalin-fixed paraffin-embedded tissues; Intraepithelial neoplasia
The aim of this project was to develop a method to assess fiber type specific protein content across the continuum of human skeletal muscle fibers. Individual vastus lateralis muscle fibers (n = 264) were clipped into two portions, one for SDS-PAGE fiber typing and one for Western blot protein identification. Following fiber type determination, fiber segments were combined into fiber type specific pools (~20 fibers/pool) and measured for total protein quantity, GAPDH, citrate synthase, and total p38 content. GAPDH content was 64%, 54%, 160%, and 138% more abundant in MHC I/IIa, MHC IIa, MHC IIa/IIx, and MHC IIx fibers when compared to MHC I. Inversely, citrate synthase content was 528%, 472%, 242%, and 47% more abundant in MHC I, MHC I/IIa, MHC IIa, and MHC IIa/IIx fibers when compared to MHC IIx. Total p38 content was 87% greater in MHC IIa versus MHC I fibers. These data and approach establish a reliable method for human skeletal muscle fiber type specific protein analysis. Initial results show particular proteins exist in a hierarchal fashion throughout the continuum of human skeletal muscle fiber types, further highlighting the necessity of fiber type specific analysis.
Western blot; GAPDH; Citrate Synthase; p38
Glycosyltransferases (GlycoTs) catalyze the transfer of monosaccharides from nucleotide-sugars to carbohydrate, lipid and protein based acceptors. We examined strategies to scale-down and increase the throughput of glycoT enzymatic assays since traditional methods require large reaction volumes and complex chromatography. Approaches tested utilized: i) Microarray pin-printing. This method was appropriate when glycoT activity was high; ii) Microwells and microcentrifuge tubes. This was suitable for studies with cell lysates when enzyme activity was moderate; iii) C18 pipette tips and solvent-extraction. This enriched reaction product when the extent of reaction was low. In all cases, reverse phase-thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrate an increase in overall β(1,3)galactosyltransferase and α(2,3)sialyltransferase activity, and a decrease in α(1,3)fucosyltransferases when these cells differentiation towards cardiomyocytes. Enzymatic and lectin binding data suggest a transition from LeX type structures in mESCs to sialylated Galβ1,3GalNAc type glycans upon differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated to cardiomyocytes. Overall, simple, rapid, quantitative and scalable glycoT activity analysis methods are presented. These utilize a range of natural and synthetic acceptors for the analysis of complex biological specimen that have limited availability.
embryonic stem cells; cardiomyocytes; glycan; radioactivity; lectins; glycosyltransferase