Affinity Purification Method Research Articles

Early Life Stress Restricts Translational Reactivity in CA3 Neurons Associated With Altered Stress Responses in Adulthood.

Early life experiences program brain structure and function and contribute to behavioral endophenotypes in adulthood. Epigenetic control of gene expression by those experiences affect discrete brain regions involved in mood, cognitive function and regulation of hypothalamic-pituitary-adrenal (HPA) axis. In rodents, acute restraint stress increases the expression of the repressive histone H3 lysine 9 tri-methylation (H3K9me3) in hippocampal fields, including the CA3 pyramidal neurons. These CA3 neurons are crucially involved in cognitive function and mood regulation as well as activation of glucocorticoid (CORT) secretion. CA3 neurons also exhibit structural and functional changes after early-life stress (ELS) as well as after chronic stress in adulthood. Using a protocol of chronic ELS induced by limited bedding and nesting material followed by acute-swim stress (AS) in adulthood, we show that mice with a history of ELS display a blunted CORT response to AS, despite exhibiting activation of immediate early genes after stress similar to that found in control mice. We find that ELS induced persistently increased expression of the repressive H3K9me3 histone mark in the CA3 subfield at baseline that was subsequently decreased following AS. In contrast, AS induced a transient increase of this mark in control mice. Using translating ribosome affinity purification (TRAP) method to isolate CA3 translating mRNAs, we found that expression of genes of the epigenetic gene family, GABA/glutamate family, and glucocorticoid receptors binding genes were decreased transiently in control mice by AS and showed a persistent reduction in ELS mice. In most cases, AS in ELS mice did not induce gene expression changes. A stringent filtering of genes affected by AS in control and ELS mice revealed a noteworthy decrease in gene expression change in ELS mice compared to control. Only 18 genes were selectively regulated by AS in ELS mice and encompassed pathways such as circadian rhythm, inflammatory response, opioid receptors, and more genes included in the glucocorticoid receptor binding family. Thus, ELS programs a restricted translational response to stress in stress-sensitive CA3 neurons leading to persistent changes in gene expression, some of which mimic the transient effects of AS in control mice, while leaving in operation the immediate early gene response to AS.

An altered miTRAP method for miRNA affinity purification with its pros and cons.

The major mechanisms of posttranscriptional gene regulation involve microRNAs (miRs) and RNA-binding proteins (RBPs). Recent studies not only identified functionally and characterized such factors, but rather investigated their use as biomarkers and suitability as biopharmaceuticals. Indeed, some miR-based drugs are currently tested in clinical studies as potential anti-viral and as anti-cancer agents. For the chemical application, a profound knowledge of the binding affinities of miRs and RBPs to their target RNA is essential. The authors recently identified several miRs regulating the non-classical human leukocyte antigen (HLA)-G, and characterized their binding affinity to the 3' untranslated region (UTR) of HLA-G. These miRs identified by miTRAP were classified into high affinity and low affinity miRs, which were either key regulators or fine tuners of HLA-G. While the miTRAP method has been described in detail, a novel modified miTRAP technique has been established, which completely consists of commercially available components and uses a simplified cloning strategy. This technique allows the identification and characterization of miRs and RBPs for any RNA sequence of interest.

250-LB: Affinity Purification and Identification of Unique, Shared, Anti-IA-2 B-Cell Clonotypes: A Proteomic Approach to Understanding Pathophysiological Mechanisms Underpinning Humoral Autoimmunity in Type 1 Diabetes

Immune destruction of beta (β) cells is a feature of type 1 diabetes (T1D), yet the pathophysiological role of autoantibodies (Abs) is poorly understood. Recently, proteomic interrogation of Abs indicates that restricted heavy (H) and light (L) chain gene usage underpins targeting of classic autoantigens in systemic autoimmunity. In the current study, we have taken a similar approach to characterise B cell clonotypes resulting in targeting of the major T1D autoantigen, IA-2. Firstly, we developed a novel ELISA assay for the major IA-2 epitopes using an MBP-fusion protein (FP) incorporating intracellular domain of IA-2. We next established ELISA-based affinity purification (AP) method using the IA-2-MBP FP to isolate enriched IA-2 specific Abs from 2 patients with verified (by RSR commercial ELISA) anti-IA-2 autoimmunity. The AP IA-2 Abs were then analysed for Ig H and L chain clonality, variable region gene usage and AA sequence using high-res electrophoretic fractionation and de-novo/database-driven analysis of AA peptides (TripleTOF 5600+ MS). We report the presence of 2 unique Ig Kappa-restricted clonotypes giving rise to IA-2 autoimmunity; namely a IGHV3.23 H chain paired with either a IGKV1.9 or IGKV3.20 L chain. Remarkably, both clones were present in AP Ig from 2 unrelated T1D patients, with shared AA replacement mutations. The Ig signatures were not present in healthy, age/sex matched controls, T1D patients negative for anti-IA-2, or Lupus. These extraordinary findings suggest the pathogenic mechanisms leading to IA-2 autoimmunity may be virtually identical across individuals, leading to the intriguing possibility of intervention in the processes underpinning the disease. Furthermore, the ability to AP and identify genuine human clonal IA-2 Abs will unlock functional studies aimed at establishing the role of these Abs in the pathophysiology of β cell loss. Disclosure T. Mendis: None. J. Wang: None. M. Pietropaolo: None. M.W. Jackson: None.

The cardiac syndecan-4 interactome reveals a role for syndecan-4 in nuclear translocation of muscle LIM protein (MLP)

Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart.

Development of GEM-PA-nanotrap for purification of foot-and-mouth disease virus

Vaccination is the primary preventative measure against outbreaks of foot-and-mouth disease (FMD). The efficacy of inactivated FMD vaccines is mainly determined by the integrity of foot-and-mouth disease virus (FMDV) particles (referred to as 146S particles), and impurities in the inactivated vaccines could result in side effects. In this study, we developed an effective affinity purification method for the purification of FMDV from cellular lysates, referred to as GEM-PA-nanotrap. To develop the GEM-PA-nanotrap, a nanobody (Nb205) against FMDV vaccine strain O/MYA98/BY/2010 146S particles was selected from a non-immunized library and fused to a peptidoglycan-binding protein anchor (PA). The PA-Nb205 fusion protein was non-covalently coupled to the surface of Gram-positive enhancer matrix (GEM) particles, which were prepared from the non-living, non-genetically modified, Gram-positive, food-grade Lactococcus lactis bacteria. The GEM-PA-nanotrap was used to purify FMDV from cellular lysates through a simple incubation and centrifugation step. The FMDV recovery rate was more than 99%, the efficiency of nonviral protein removal was about 98.3%, and the purification process had almost no effect on the integrity and immunogenicity of 146S particles. Therefore, the GEM-PA-nanotrap has potential as an effective method for the recovery and purification of FMDV during the vaccine manufacturing process.

Proteomic navigation using proximity-labeling.

The identification of bona fide protein-protein interactions and the mapping of proteomes was greatly enhanced by protein tagging for generic affinity purification methods and analysis by mass spectrometry (AP-MS). The high quality of AP-MS data permitted the development of proteomic navigation by sequential tagging of identified interactions. However AP-MS is laborious and limited to relatively high affinity protein-protein interactions. Proximity labeling, first with the biotin ligase BirA, termed BioID, and then with ascorbate peroxidase, termed APEX, permits a greater reach into the proteome than AP-MS enabling both the identification of a wider field and weaker protein-protein interactions. This additional reach comes with the need for stringent controls. Proximity labeling also permits experiments in living cells allowing spatiotemporal investigations of the proteome. Here we discuss proximity labeling with accompanying methodological descriptions for E. coli and mammalian cells.

Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag

The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3′-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.

An Easy and Sensitive Method to Profile the Antibody Specificities of HLA-specific Memory B Cells.

Pretransplant immunological risk assessment is currently based on donor-specific HLA antibodies in serum. Despite being an excellent source for antibodies produced by bone marrow-residing plasma cells, serum analysis does not provide information on the memory B-cell compartment. Although B-cell culture supernatants can be used to detect memory B cell-derived HLA antibodies, low IgG concentrations can preclude detectability of HLA antibodies in luminex single-antigen bead (SAB) assays. Culture supernatants of polyclonally activated B cells from alloantigen exposed (n = 13) or nonexposed (n = 10) individuals were either concentrated 10-fold, or IgG was isolated by using a protein G affinity purification method to increase the IgG concentration. These processed culture supernatants, as well as paired serum samples were tested for the presence of HLA antibodies using luminex SAB analysis. In immunized individuals, 64% were found to have HLA-specific B-cell memory in concentrated supernatants, whereas 82% showed HLA-specific B-cell memory when IgG isolated supernatants were used for HLA antibody detection. IgG-isolated supernatants showed higher mean fluorescence intensity values compared with concentrated supernatants without increased background. In some individuals, HLA-specific B-cell memory was detected in the absence of accompanying serum antibody specificities. We developed a novel, highly sensitive method to assess the HLA-specific memory B-cell compartment using luminex SAB technology. This assay allows direct comparison to the serum compartment and may therefore provide a more complete picture of the humoral alloimmune response in patients with a history of alloantigen exposure.

Selective Identification and Site-Specific Quantification of 4-Hydroxy-2-nonenal-Modified Proteins.

4-Hydroxy-2-nonenal (HNE)-modified proteins are closely associated with cellular functions and diseases, so qualitative and quantitative analysis of HNE-modified proteins is very necessary in order to further understand their structures and molecular functions. In this study, we described a six-plex isobaric labeling affinity purification (SiLAP) method based on the interaction of aminoxyTMT six-plex and anti-TMT antibody resin to identify and quantify the HNE modifications simultaneously. The labeling efficiency, ionization efficiency of the aminoxyTMT-tagged peptides, and reliability of the quantification method were investigated in detail. The mass tags were labeled on the modification sites, which could also significantly increase the ionization efficiency, contributing to site-specific identification and quantification of HNE peptides. The SiLAP strategy possessed high sensitivity, accuracy, and good reproducibility to qualitatively and quantitatively analyze HNE-modified proteins/peptides, which could be used to analyze both endogenously and exogenously modified proteins. Using the SiLAP strategy, 2257 HNE-modified peptides mapping 1121 proteins were collectively quantified, which was the largest data set of HNE-modified proteins with detailed modification sites, and 101 proteins were found to be differentially modified by HNE in six liver cell lines. At the same time, 33 endogenously HNE-modified peptides mapping 33 proteins were identified with modification sites.

Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome.

Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins.

Integrated Succinylome and Metabolome Profiling Reveals Crucial Role of S-Ribosylhomocysteine Lyase in Quorum Sensing and Metabolism of Aeromonas hydrophila*[S

Protein modification by lysine succinylation is a newly identified post-translational modification (PTM) of lysine residues and plays an important role in diverse physiological functions, although their associated biological characteristics are still largely unknown. Here, we investigated the effects of lysine succinylation on the physiological regulation within a well-known fish pathogen, Aeromonas hydrophila A high affinity purification method was used to enrich peptides with lysine succinylation in A. hydrophila ATCC 7966, and a total of 2,174 lysine succinylation sites were identified on 666 proteins using LC-MS/MS. Gene ontology analysis indicated that these succinylated proteins are involved in diverse metabolic pathways and biological processes, including translation, protein export, and central metabolic pathways. The modifications of several selected candidates were further validated by Western blotting. Using site-directed mutagenesis, we observed that the succinylation of lysines on S-ribosylhomocysteine lyase (LuxS) at the K23 and K30 sites positively regulate the production of the quorum sensing autoinducer AI-2, and that these PTMs ultimately alter its competitiveness with another pathogen, Vibrio alginolyticus Moreover, subsequent metabolomic analyses indicated that K30 succinylation on LuxS may suppress the activated methyl cycle (AMC) and that both the K23 and K30 sites are involved in amino acid metabolism. Taken together, the results from this study provide significant insights into the functions of lysine succinylation and its critical roles on LuxS in regulating the cellular physiology of A. hydrophila.

Design and Synthesis of a Chitodisaccharide-Based Affinity Resin for Chitosanases Purification.

Chitooligosaccharides (CHOS) have gained increasing attention because of their important biological activities. Enhancing the efficiency of CHOS production essentially requires screening of novel chitosanase with unique characteristics. Therefore, a rapid and efficient one-step affinity purification procedure plays important roles in screening native chitosanases. In this study, we report the design and synthesis of affinity resin for efficient purification of native chitosanases without any tags, using chitodisaccharides (CHDS) as an affinity ligand, to couple with Sepharose 6B via a spacer, cyanuric chloride. Based on the CHDS-modified affinity resin, a one-step affinity purification method was developed and optimized, and then applied to purify three typical glycoside hydrolase (GH) families: 46, 75, and 80 chitosanase. The three purified chitosanases were homogeneous with purities of greater than 95% and bioactivity recovery of more than 40%. Moreover, we also developed a rapid and efficient affinity purification procedure, in which tag-free chitosanase could be directly purified from supernatant of bacterial culture. The purified chitosanases samples using such a procedure had apparent homogeneity, with more than 90% purity and 10–50% yield. The novel purification methods established in this work can be applied to purify native chitosanases in various scales, such as laboratory and industrial scales.

Current affinity approaches for purification of recombinant proteins

Recombinant proteins have wide applications in the development of pharmaceutical compounds, industrial applications of enzymes, and basic proteomics research. In this way, efficient production of recombinant proteins with high purity needs efficient purification methods. Various strategies have been devised to improve these proteins purification such as affinity purification and physicochemical purification methods which the affinity purification has some advantages over the others. Affinity strategies especially fusion strategies have been devised as indispensable tools for the massive parallel production, identification and purification of recombinant proteins from the host systems. These strategies facilitate commercial and industrial formulations of recombinant proteins, improve the study of protein interactions at the molecular level, and develop highly sensitive and specific bioassays. Recently, various surface-modified nanoparticles have been widely developed to enhance recovery and purification of recombinant proteins such as Hydrophobic polymer nanoparticles and Oleosin nanoparticles. In this review, we aim to discuss affinity purification technologies and address the principles, advantages, limitations and potential applications of them.

Tandem Affinity Purification of SBP-CBP-tagged Type Three Secretion System Effectors.

Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.

Identification of Immune Modulatory miRNAs by miRNA Enrichment via RNA Affinity Purification.

Immune escape by cancer cells can be triggered by aberrant expression of immunological key players, which can be achieved by distinct molecular mechanisms including immune modulatory miRNAs. One suitable method to identify miRNAs that specifically target immune relevant molecules is the miRNA enrichment via RNA affinity purification method named miTRAP (miRNA trapping by RNA in vitro affinity purification). Here, we present a detailed protocol for construct preparation, RNA immobilization via MS2BP-MBP to beads, miRNA enrichment, and elution followed by analysis of the obtained miRNA candidates via qRT-PCR.

Two-Step Co-Immunoprecipitation (TIP).

In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein-protein complex from Burkitt lymphoma cells and from primary human CD4+ T cells. In addition, this unit describes an application of TIP for the isolation of transcription-factor-bound chromatin. © 2018 by John Wiley & Sons, Inc.

A novel magnetic beads-based method for polioviral concentration from environmental samples

Environmental surveillance (EnvS)22Environmental surveillance (ENVS). is an important tool for monitoring the presence of poliovirus in endemic and poliovirus free regions. Unlike acute flaccid paralysis (AFP)33Acute flaccid paralysis (AFP). surveillance, EnvS can monitor large populations using small numbers of samples and detect the introduction of poliovirus even before the appearance of AFP cases. Early detection and timely response can prevent the onset of poliovirus associated AFP, as was demonstrated by silent poliovirus transmission in Israel in 2013. Although EnvS is currently recommended as supplementary to AFP surveillance, it is limited to laboratories with equipment for poliovirus concentration and to regions where samples can be easily transported under temperature controlled conditions to such facilities. However the highest risk of poliovirus re-emergence is in developing countries where such conditions do not exist. We developed and evaluated an affinity purification method using antibody or poliovirus receptor (CD155) presenting bacteriophage covered magnetic beads for poliovirus concentration. This method requires only simple, inexpensive and portable equipment. Though tested only on Sabin 1 spiked sewage samples it provided better recovery than our current polyethylene glycol (PEG)44Polyethylene glycol (PEG)./NaCl- based concentration method. On site use of this method might facilitate EnvS in currently inaccessible remote regions by significantly reducing the volume of sample that needs to be transported back to the laboratory under temperature-controlled conditions554°C..

RIG-I Recognizes the 5′ Region of Dengue and Zika Virus Genomes

The flavivirus genus comprises major human pathogens, such as Dengue (DENV) and Zika (ZIKV) viruses. RIG-I and MDA5 are key cytoplasmic pathogen recognition receptors that are implicated in detecting viral RNAs. Here, we show that RNAs that co-purified with RIG-I during DENV infection are immuno-stimulatory, whereas RNAs bound to MDA5 are not. An affinity purification method combined with next-generation sequencing (NGS) revealed that the 5' region of the DENV genome is recognized by RIG-I. No DENV RNA was bound to MDA5. Invitroproduction of fragments of the DENV genome confirmed the NGS data and revealed that the 5'end of the genome, when bearing 5'-triphosphates, is the RIG-I ligand. The 5' region of the ZIKV genome is also a RIG-I agonist. We propose that RIG-I binds to the highly structured and conserved 5' region of flavivirus nascent transcripts before capping and that this mechanism leads to interferon secretion by infected cells.

IASPP\u2013PP1 complex is required for cytokinetic abscission by controlling CEP55 dephosphorylation

Cytokinesis is the last step of cell division and is concluded by the abscission of the intercellular bridge that connects two daughter cells. The tight regulation of cytokinesis completion is essential because cytokinesis failure is associated with various human diseases. Here, we report that iASPP, a member of the apoptosis-stimulating proteins of p53 (ASPP) family, is required for proper cell division. iASPP depletion results in abnormal midbody structure and failed cytokinesis. We used protein affinity purification methods to identify the functional partners of iASPP. We found that iASPP associates with centrosomal protein of 55 kDa (CEP55), an important cytokinetic abscission regulator. Mechanically, iASPP acts as a PP1-targeting subunit to facilitate the interaction between PP1 and CEP55 and to remove PLK1-mediated Ser436 phosphorylation in CEP55 during late mitosis. The latter step is critical for the timely recruitment of CEP55 to the midbody. The present observations revealed a previously unrecognized function of iASPP in cytokinesis. This function, in turn, likely contributes to the roles of iASPP in tumor development and genetic diseases.

UvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins

RNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation, and stability of mRNAs. We describe ultraviolet crosslinking and affinity purification (uvCLAP), an easy-to-use, robust, reproducible, and high-throughput method to determine in vivo targets of RBPs. uvCLAP is fast and does not rely on radioactive labeling of RNA. We investigate binding of 15 RBPs from fly, mouse, and human cells to test the method’s performance and applicability. Multiplexing of signal and control libraries enables straightforward comparison of samples. Experiments for most proteins achieve high enrichment of signal over background. A point mutation and a natural splice isoform that change the RBP subcellular localization dramatically alter target selection without changing the targeted RNA motif, showing that compartmentalization of RBPs can be used as an elegant means to generate RNA target specificity.