Purification Of Streptavidin Research Articles

Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Using BioID for the Identification of Interacting and Proximal Proteins in Subcellular Compartments in Toxoplasma gondii.

BioID is an in vivo biotinylation system developed to examine the proximal and interacting proteins of a bait protein within a subcellular compartment. This approach has been exploited in Toxoplasma for protein-protein interaction studies and proteomic characterizations of intracellular compartments. The BioID method requires constructing a translational fusion between a protein of interest and the promiscuous biotin ligase BirA∗ (a mutant of the E. coli protein BirA) which enables trafficking of the protein to the correct intracellular compartment and association with its partners. Proximity labelling occurs upon addition of biotin to the media and the biotinylated target proteins are then be purified using stringent conditions via streptavidin chromatography. In this chapter, we describe the methodology to fuse BirA∗(or the newer variant BioID2) to a bait protein using endogenous gene tagging in Toxoplasma and then identify the proximal and interacting proteins using in vivo biotinylation, streptavidin purification and mass spectrometric analysis.

Identification of AcMNPV GP64-binding proteins through a combinational use of a self-biotinylated virus and the cross-linking method

Baculoviruses are potential vectors of gene therapy for the ability to transfer gene high efficiently into mammalian cells. However, cell membrane proteins which interact with baculoviral glycoproteins have not been identified. In this study, we developed a self-biotinylated AcMNPV bearing biotinylated GP64 glycoproteins. This recombinant virus demonstrated the capability to infect insect cells and to transduct mammalian cells. Using this biotinylated virus, a protein >170Kda which could specifically interact with GP64 proteins was identified from virus transducted BHK-21 cells through cross-linking and streptavidin purification. Our study provides a useful approach for identifying cell membrane proteins that interact with baculovirus surface proteins or proteins involved in virus attachment.

Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal.

Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue–residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.

Production and purification of streptavidin with higher biotin-binding activity

The objective of this study was to develop practical, efficient method for production, purification and assay of binding activity of streptavidin. Streptomyces avidinii was first propagated on agar plates, the bacterial cells on the agar were scrapped and suspended in a defined synthetic media (4.4 ml/cm 2 ). After 7 days agitation on a rotary shaker (200 rpm/min) at room temprature (≈28°C), the bacterial cells in the culture were pelleted. The culture supernatant was concentrated to 1/62 original volume with 75% saturation ammonium sulphate. After intensive dialysis against ammonium carbonate buffer pH 11, the suspension was loaded into an iminobiotin agarose column chromatography. The adsorbed protein (streptavidin) was eluted with sodium acetate buffer, pH 4, and the eluate was concentrated with an ultrafiltration divice and suspended in PBS. The strepatavidin-binding activity was assayed by a competitive ELISA, a competition between streptavidin in the sample and the HRP-streptavidin conjugate for the biotin (biotinyl IgG) immobilised on wells of a microtitre plate. The detection limit of this assay measured 0.16 µg/ml streptavidin. The method developed in this study produced 160 µg/ml streptavidin in the culture supernatant. After concentration with the ammonium sulphate, the streptavidin concentration increased to 4 mg/ml (69% recovery). At the final step of purification, streptavidin with 10 mg/ml concentration was obtained. The purity of the streptavidin was higher (95%) with a recovary of 19%. The purified streptavidin in this study appeared as a dimer core streotavidin on SDS PAGE and its binding activity was twice as high as that of a commercial one.

Conserved ion and amino acid transporters identified as phosphorylcholine-modified N-glycoproteins by metabolic labeling with propargylcholine in Caenorhabditis elegans cells.

Phosphorylcholine (PC) modification of proteins by pathogens has been implicated in mediating host-pathogen interactions. Parasitic nematodes synthesize PC-modified biomolecules that can modulate the host's antibody and cytokine production to favor nematode survival, contributing to long-term infections. Only two nematode PC-modified proteins (PC-proteins) have been unequivocally identified, yet discovering the protein targets of PC modification will be paramount to understanding the role(s) that this epitope plays in nematode biology. A major hurdle in the field has been the lack of techniques for selective purification of PC-proteins. The nonparasitic nematode Caenorhabditis elegans expresses PC-modified N-linked glycans, offering an attractive model to study the biology of PC-modification. We developed a robust method to identify PC-proteins by metabolic labeling of primary embryonic C. elegans cells with propargylcholine, an alkyne-modified choline analog. Cu(I)-catalyzed cycloaddition with biotin-azide enables streptavidin purification and subsequent high-throughput LC-MS identification of propargyl-labeled proteins. All proteins identified using stringent criteria are known or predicted to be membrane or secreted proteins, consistent with the model of a Golgi-resident, putative PC-transferase. Of the 55 PC-N-glycosylation sites reported, 33 have been previously observed as N-glycosylation sites in high-throughput screens of C. elegans. Several identified PC-proteins are nematode-specific proteins, but 10 of the PC-proteins are widely conserved ion transporters and amino acid transporters, while eight are conserved proteins involved in synaptic function. This finding suggests a functional role for PC-modification beyond immunomodulation. The approach presented in this study provides a method to identify PC-proteins in C. elegans and related nematodes.

Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.

Proteomic Analysis of Two Metabolic Proteins with Potential to Translocate to Plasma Membrane Associated with Tumor Metastasis Development and Drug Targets

Metastasis is the main cause for death of breast cancer patients. However, the underlying mechanism is still poorly understood. Plasma membrane (PM) proteins play a key role in various biological processes, especially for cell migration. In this study, we used a set of well-characterized mammary mouse cell lines, 67NR, 168FARN, 4T1, representing the metastatic progression, to study the differentially expressed membrane proteins. These proteins were analyzed by a linear ion trap tandem mass spectrometry (LTQ-MS/MS) following cell surface biotinylation and streptavidin purification. A total of 1667 membrane proteins were identified, out of which 472 were characterized as differentially expressed with at least 2-fold change and p-value < 0.01. Functional clustering of the 472 proteins revealed that 178 of them were metabolic proteins. Finally, we focused on two metabolic proteins, fatty acid synthase (FASN) and NAD(P)H steroid dehydrogenase-like protein (NSDHL), which were validated by Western blot and immunofluorescence. We found that FASN and NSDHL translocated to the plasma membrane from the intracellular compartment, and their expressions increased from 67NR to 4T1. This alteration of localization along with differential expressions might be necessary for metastasis development. Potentially, FASN and NSDHL could serve as drug targets in new antimetastasis therapy.

Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA

Understanding the processes of DNA replication, chromatin assembly and maturation, and the replication stress response requires the ability to monitor protein dynamics at active and damaged replication forks. Detecting protein accumulation at replication forks or damaged sites has primarily relied on immunofluorescence imaging, which is limited in resolution and antibody sensitivity. Here we describe a procedure to isolate proteins on nascent DNA (iPOND) that permits a high-resolution spatiotemporal analysis of proteins at replication forks or on chromatin following DNA replication in cultured cells. iPOND relies on labeling of nascent DNA with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). Biotin conjugation to EdU-labeled DNA using click chemistry facilitates a single-step streptavidin purification of proteins bound to the nascent DNA. iPOND permits an interrogation of any cellular process linked to DNA synthesis using a 3- to 4-d protocol.

Development and mapping of SNP assays in allotetraploid cotton

A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin–streptavidin purification, and agarose gel separation) on two accessions of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2.04 million genomic sequence reads were assembled into contigs with an N50 of 508 bp and analyzed for SNPs. A previously generated assembly of expressed sequence tags (ESTs) provided an additional source for SNP discovery. Using highly conservative parameters (minimum coverage of 8× at each SNP and 20% minor allele frequency), a total of 11,834 and 1,679 non-genic SNPs were identified between accessions of G. hirsutum and G. barbadense in genome reduction assemblies, respectively. An additional 4,327 genic SNPs were also identified between accessions of G. hirsutum in the EST assembly. KBioscience KASPar assays were designed for a portion of the intra-specific G. hirsutum SNPs. From 704 non-genic and 348 genic markers developed, a total of 367 (267 non-genic, 100 genic) mapped in a segregating F2 population (Acala Maxxa × TX2094) using the Fluidigm EP1 system. A G. hirsutum genetic linkage map of 1,688 cM was constructed based entirely on these new SNP markers. Of the genic-based SNPs, we were able to identify within which genome (‘A’ or ‘D’) each SNP resided using diploid species sequence data. Genetic maps generated by these newly identified markers are being used to locate quantitative, economically important regions within the cotton genome.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-011-1780-8) contains supplementary material, which is available to authorized users.

Immobilized Iminobiotin on Magnetic Poly (Vinyl Alcohol) Microspheres for Single-step Purification of Streptavidin

Magnetic poly (vinyl alcohol) microspheres (MPVAMS) immobilized with iminobiotin as the ligand were prepared for the purification of streptavidin. Parameters of magnetic separation, including spacers, NHS-iminobiotin added, elution behavior, and sample treatment, were optimized to improve the purification efficiency to achieve the maximum recovery of the protein. Streptavidin was successfully purified 38-fold from culture supernatant in a single-step by iminobiotin-MPVAMS with an overall recovery of 90.15% and purity of 95.08%. Hence, this study effectively illustrated the favorable application of magnetic microcarriers for the purification of streptavidin.

Affinity purification of streptavidin using tobacco mosaic virus particles as purification tags

A new protein affinity purification system has been developed. Recombinant tobacco mosaic virus (TMV) was used as an affinity matrix for isolation and purification of the given protein of interest. In model experiments, streptavidin-specific heptapeptide sequence TLIAHPQ was inserted into TMV coat protein near the C end. This oligopeptide did not interfere significantly with viral replication, assembly, and movement. Recombinant TMV functioned as an epitope tag recognizing streptavidin in plant protein extracts. Plant protein extracts containing streptavidin were incubated with recombinant TMV virions. Affinity complexes of viral particles with the protein of interest were collected by centrifugation. Recombinant TMV–streptavidin complex was dissociated with 0.2 M acetic acid, pH 4.6, and was passed through membrane filter Nanosep 300 K by centrifugation. The filtrate contained pure streptavidin. Recombinant TMV was left on the filter. TMV particles collected from the filter could be used for at least two more purification cycles. The streptavidin-specific recombinant TMV system was applied successfully for purification of streptavidin from Streptomyces avidinii. The authors believe that the TMV-based affinity system can also be used for the purification of other proteins.

Procedure for the purification of streptavidin by hydrophobic interaction chromatography

A procedure is described for the purification of hydrophobic microbial proteins such as streptavidin from Streptomyces avidinii, using Benzyl-DC bead cellulose as the column material. The separation is rapid with a high loading capacity and sufficient resolution for preparative uses. Advantages are discussed especially for industrial purposes.

Improvement of production, assay and purification of streptavidin.

The production of streptavidin by Streptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay. This assay appears to give useful estimates of streptavidin production. Depending upon the medium employed, streptavidin yields ranged from 0.5 mg/l to 53 mg/l. Production was successfully scaled up to ten liter fermentors. Streptavidin was purified in a one step process from centrifuged, concentrated fermentation broths by binding the protein to an iminobiotin column at pH 11 followed by elution at pH 4.0. Recovery percentages varied depending upon the solubility of the fermentation media ingredients.

Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium

A method is described for isolation of streptavidin from cultures of Streptomyces avidinii grown in a synthetic culture medium for 6–10 days. Streptavidin is precipitated directly from culture supernatant fluid using 80% ammonium sulfate, and the precipitate is dialyzed against water and centrifuged at 40 000 × g for 60 min. The absorbancy coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 ± 0.1806 for 1% solution. The protein appeared to be >90% homogenous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules <70 kDa in size were detected at any step during the purification of streptavidin. Streptavidin was able to maintain a stable crosslink between two biotinylated molecules in a solid-phase assay. Streptavidin purified by this method was stable in 50% glycerol/water at −20°C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.

Production of streptavidin in a synthetic medium

A simple, inexpensive procedure for producing streptavidin has been described. The biotin-binding protein was produced by growing Streptomyces avidinii in a synthetic liquid culture medium containing l-asparagine as the sole nitrogen source. With this procedure, extraneous proteinaceous substances inherently present in culture media prepared with yeast extract or with peptones were not present to interfere with isolation and purification of streptavidin. When harvested after 7–8 days of incubation, the culture fluid was relatively free of contaminating cell breakdown products. Maximal production of streptavidin (100–120 mg/1) was obtained in 8–10 day cultures. For some applications, the culture fluid can be used directly as a source of streptavidin. Under the same conditions used to grow S. avidinii, 11 other actinomycete strains and 134 eumycetes were found to lack the capacity to produce detectable amounts of an extracellular biotin-binding protein.

An improved method for the single-step purification of streptavidin

A new method for the preparation of a more efficient, stable iminobiotin-containing resin for the isolation of streptavidin was developed. CL-Sepharose was activated with p-nitrophenyl chloroformate, and the resultant carbonate derivative was reacted with diaminohexane. Subsequent reaction of the amino-containing resin with iminobiotin- N-hydroxysuccinimide ester (in an organic solvent) yielded the stable affinity resin. The capacity of this resin for either avidin of streptavidin was 12 mg per ml resin, and streptavidin could be purified in one step directly from the culture broth of Streptomyces avidinii. The biotin-binding protein isolated in this manner exhibited a major band at about 75 kDa and a minor band at about 150 kDa. Under denaturing conditions, a spectrum of subunit molecular weights ranging between 15 and 19 kDa was detected, the distributiob of which depended upon the specific preparation.