Search tips
Search criteria

Results 1-25 (942989)

Clipboard (0)

Related Articles

1.  Application of magnetic techniques in the field of drug discovery and biomedicine 
Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.
PMCID: PMC212320  PMID: 14521720
2.  Streamlined protein expression and purification using cleavable self-aggregating tags 
Recombinant protein expression and purification remains a fundamental issue for biotechnology. Recently we found that two short self-assembling amphipathic peptides 18A (EWLKAFYEKVLEKLKELF) and ELK16 (LELELKLKLELELKLK) can induce the formation of active protein aggregates in Escherichia coli (E. coli), in which the target proteins retain high enzymatic activities. Here we further explore this finding to develop a novel, facile, matrix-free protein expression and purification approach.
In this paper, we describe a streamlined protein expression and purification approach by using cleavable self-aggregating tags comprising of one amphipathic peptide (18A or ELK16) and an intein molecule. In such a scheme, a target protein is first expressed as active protein aggregate, separated by simple centrifugation, and then released into solution by intein-mediated cleavage. Three target proteins including lipase A, amadoriase II and β-xylosidase were used to demonstrate the feasibility of this approach. All the target proteins released after cleavage were highly active and pure (over 90% in the case of intein-ELK16 fusions). The yields were in the range of 1.6-10.4 μg/mg wet cell pellet at small laboratory scale, which is comparable with the typical yields from the classical his-tag purification, the IMPACT-CN system (New England Biolabs, Beverly, MA), and the ELP tag purification scheme.
This tested single step purification is capable of producing proteins with high quantity and purity. It can greatly reduce the cost and time, and thus provides application potentials for both industrial scale up and laboratorial usage.
PMCID: PMC3124420  PMID: 21631955
3.  Characterization of magnetic nanoparticles using programmed quadrupole magnetic field-flow fractionation 
Quadrupole magnetic field-flow fractionation is a relatively new technique for the separation and characterization of magnetic nanoparticles. Magnetic nanoparticles are often of composite nature having a magnetic component, which may be a very finely divided material, and a polymeric or other material coating that incorporates this magnetic material and stabilizes the particles in suspension. There may be other components such as antibodies on the surface for specific binding to biological cells, or chemotherapeutic drugs for magnetic drug delivery. Magnetic field-flow fractionation (MgFFF) has the potential for determining the distribution of the magnetic material among the particles in a given sample. MgFFF differs from most other forms of field-flow fractionation in that the magnetic field that brings about particle separation induces magnetic dipole moments in the nanoparticles, and these potentially can interact with one another and perturb the separation. This aspect is examined in the present work. Samples of magnetic nanoparticles were analysed under different experimental conditions to determine the sensitivity of the method to variation of conditions. The results are shown to be consistent and insensitive to conditions, although magnetite content appeared to be somewhat higher than expected.
PMCID: PMC2981903  PMID: 20732895
magnetic nanoparticles; field-flow fractionation; characterization; quadrupole magnet; magnetic field-flow fractionation; magnetic dipole interaction
4.  Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS) 
Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.
PMCID: PMC3144656  PMID: 20644514
5.  P176-M Rapid Purification and Enrichment of Proteins from Crude Solutions Using Reversed Phase Microparticles and Subsequent Generation of Peptide Mass Fingerprints by MALDI-TOF Mass Spectrometry 
The generation of peptide mass fingerprints followed by a database search is a common tool for the mass spectrometric identification of proteins. To provide a high sensitivity, proteins must be efficiently purified and concentrated prior to enzymatic digestion. Common desalting procedures, like ultrafiltration or dialysis, are very time consuming and work best for high protein concentrations. Another critical point is the proteolysis of the investigated protein, which works efficiently only with concentrated protein solutions. Therefore, efficient concentration and simultaneous purification using solid phase extraction (SPE) will be the method of choice to receive pure and highly concentrated protein solutions prior to enzymatic digestion. In this work, we manufactured magnetic reversed phase particles for the efficient purification and simultaneous concentration of protein samples with volumes up to several millilitres. The SPE procedure was compared with dialysis using commercial available microconcentrators with a cut-off membrane. Due to the magnetic core, each washing and elution step could be performed within 15 minutes. Then, the bound protein was digested directly on the beads, resulting in a remarkable increase of protein detection and better mass structural analysis. Useful MOWSE scores were achieved using bovine serum albumin as a model protein with concentrations as low as 50 ng/ml (720 pM). Compared to the dialysis procedure, which needs several hours, the isolation and purification of protein can be performed in minutes with the reversed phase particles.
PMCID: PMC2291834
6.  Magnetic DNA affinity purification of yeast transcription factor tau--a new purification principle for the ultrarapid isolation of near homogeneous factor. 
Nucleic Acids Research  1989;17(15):6253-6267.
We present a new method for rapid purification to near homogeneity of sequence specific DNA binding proteins based on magnetic separation. The method is described for the purification of the yeast transcription factor tau. DNA affinity Dynabeads (monodisperse superparamagnetic particles) specifically bind the protein in the presence of competitor DNA. By magnetic separation, wash and elution, highly enriched transcription factor preparations are obtained within minutes. In less than an hour with three cycles of adsorption, nearly homogeneous factor tau was obtained. The factor preparation contained mainly two polypeptides of 100 and 140 kDa and was fully active in transcription and DNA binding assays. This procedure should work for any high-affinity sequence-specific DNA binding protein with only minor modifications.
PMCID: PMC318276  PMID: 2671937
7.  Biological activities of histidine-rich peptides; merging biotechnology and nanomedicine 
Histidine-rich peptides are commonly used in recombinant protein production as purification tags, allowing the one-step affinity separation of the His-tagged proteins from the extracellular media or cell extracts. Genetic engineering makes feasible the post-purification His-tag removal by inserting, between the tag and the main protein body, a target site for trans-acting proteases or a self-proteolytic peptide with regulatable activities. However, for technical ease, His tags are often not removed and the fusion proteins eventually used in this form. In this commentary, we revise the powerful biological properties of histidine-rich peptides as endosomolytic agents and as architectonic tags in nanoparticle formation, for which they are exploited in drug delivery and other nanomedical applications. These activities, generally unknown to biotechnologists, can unwillingly modulate the functionality and biotechnological performance of recombinant proteins in which they remain trivially attached.
PMCID: PMC3339332  PMID: 22136342
8.  Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents 
Biotechnology and bioengineering  2011;108(8):1872-1882.
Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.
PMCID: PMC3117116  PMID: 21351074
plasmid DNA purification; non-ionic detergents; Hofmeister salts
9.  Selective Isolation of Glycoproteins and Glycopeptides for MALDI-TOF MS Detection Supported by Magnetic Particles 
Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.
PMCID: PMC2291756  PMID: 16522863
Glycosylation; Concanavalin A; boronic acid; magnetic particles; MALDI-TOF MS
10.  A Microfluidic Device for Continuous-Flow Magnetically Controlled Capture and Isolation of Microparticles 
This paper presents a novel microfluidic device that exploits magnetic manipulation for integrated capture and isolation of microparticles in continuous flow. The device, which was fabricated from poly(dimethylsiloxane) (PDMS) by soft-lithography techniques, consists of an incubator and a separator integrated on a single chip. The incubator is based on a novel scheme termed target acquisition by repetitive traversal (TART), in which surface-functionalized magnetic beads repetitively traverse a sample to seek out and capture target particles. This is accomplished by a judicious combination of a serpentine microchannel geometry and a time-invariant magnetic field. Subsequently, in the separator, the captured target particles are isolated from nontarget particles via magnetically driven fractionation in the same magnetic field. Due to the TART incubation scheme that uses a corner-free serpentine channel, the device has no dead volume and allows minimization of undesired particle or magnetic-bead retention. Single-chip integration of the TART incubator with the magnetic-fractionation separator further allows automated continuous isolation and retrieval of specific microparticles in an integrated manner that is free of manual off-chip sample incubation, as often required by alternative approaches. Experiments are conducted to characterize the individual incubation and separation components, as well as the integrated device. The device is found to allow 90% of target particles in a sample to be captured and isolated and 99% of nontarget particles to be eliminated. With this high separation efficiency, along with excellent reliability and flexibility, the device is well suited to sorting, purification, enrichment, and detection of micro/nanoparticles and cells in lab-on-a-chip systems.
PMCID: PMC3916004  PMID: 24511214
Cell sorting; magnetic manipulation; micro-fluidics; on-chip incubation; particle separation
11.  Application of Magnetic Particle Tracking Velocimetry to Quadrupole Magnetic Sorting of Porcine Pancreatic Islets 
Biotechnology and bioengineering  2011;108(9):2107-2117.
Magnetic isolation is a promising method for separating and concentrating pancreatic islets of Langerhans for transplantation in Type 1 Diabetes patients. We are developing a continuous magnetic islet sorter to overcome the restrictions of current purification methods that result in limited yield and viability. In Quadrupole Magnetic Sorting (QMS) islets are magnetized by infusing superparamagnetic microbeads into islets’ vasculature via arteries that serve the pancreas. The performance of the islet sorter depends on the resulting speed of the islets in an applied magnetic field, a property known as magnetophoretic mobility. Essential to the design and successful operation of the QMS is a method to measure the magnetophoretic mobilities of magnetically infused islets. We have adapted a Magnetic Particle Tracking Velocimeter (MPTV) to measure the magnetophoretic mobility of particles up to 1000 microns in diameter. Velocity measurements are performed in a well-characterized uniform magnetic energy gradient using video imaging followed by analysis of the video images with a computer algorithm that produces a histogram of absolute mobilities. MPTV was validated using magnetic agarose beads serving as islet surrogates and subjecting them to QMS. Mobility distributions of labeled porcine islets indicated that magnetized islets have sufficient mobility to be captured by the proposed sorting method, with this result confirmed in test isolations of magnetized islets.
PMCID: PMC3139696  PMID: 21495008
Particle tracking velocimetry; magnetic flow sorter; pancreatic islets isolation; magnetic particles
12.  Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B▿  
Applied and Environmental Microbiology  2007;73(21):6945-6952.
The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.
PMCID: PMC2074950  PMID: 17827336
13.  Synthesis and Purification of Highly Hydrophobic Peptides Derived from the C-Terminus of Amyloid β-Protein 
Some biotechnological inventions involve expensive, sophisticated machines. Others are relatively simple innovations that nevertheless address, and solve difficult problems. Synthesis and purification of highly hydrophobic peptides can be a difficult and challenging task, particularly when these peptides have low solubility in both aqueous and organic solvents. Here we describe the synthesis and purification of a series of peptides derived from the hydrophobic C-terminus of the 42-residue form of amyloid β-protein (Aβ42), a peptide believed to be the primary cause for Alzheimer’s disease (AD). The series of C-terminal fragments (CTFs) had the general formula Aβ(x-42), x=28–39, which potentially can be used as inhibitors of Aβ42 assembly and neurotoxicity. Synthesis and purification of peptides containing 8-residues or less were straightforward. However, HPLC purification of longer peptides was problematic and provided <1% yield in particularly difficult cases due to very poor solubility in the solvent systems used both in reverse- and in normal phase chromatography. Modification of the purification protocol using water precipitation followed by removal of scavengers by washing with diethyl ether circumvented the need for HPLC purification and provided these peptides with purity as high as HPLC-purified peptides and substantially increased yield.
PMCID: PMC2773559  PMID: 19898686
14.  Cell-Free Expression of Protein Kinase A for Rapid Activity Assays 
Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.
PMCID: PMC2879223  PMID: 20520741
cell-free expression; in vitro translation; HaloTag; protein immobilization; magnetic particles; PKA; kinase
15.  P149-M Proteomics Sample Preparation Using Magnetic Bead Based Technologies 
We have developed a range of magnetic beads for protein and/or peptide sample isolation and fractionation that can be utilised in proteomics strategies and workflows.
A significant challenge in proteomics today is overcoming the dynamic range of protein abundance. High throughput proteomics requires efficient methods to analyze complex protein mixtures. It is often necessary to reduce sample complexity for many proteomics strategies. The use of magnetic beads for sample preparation enables protocols to be automated and throughput to be increased. The kinetics of bead-based sample preparation is very efficient and washing can be done thoroughly. In addition, preparation is flexible regarding sample and buffer volumes. Consequently, low abundant proteins can be concentrated from large sample volumes.
We have developed protein purification methods based on the use of magnetic bead technology. These methods are used for the fractionation of complex protein samples and for isolation of proteins or peptides.
PMCID: PMC2292007
16.  Cyanogen bromide digestion of the avian myeloblastosis virus pp19 protein: isolation of an amino-terminal peptide that binds to viral RNA. 
Journal of Virology  1983;45(2):876-881.
The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.
PMCID: PMC256484  PMID: 6300441
17.  Preparative SDS-PAGE Electroelution for Rapid Purification of Alkyl Hydroperoxide Reductase from Helicobacter pylori 
Alkyl hydroperoxide reductase (AhpC) of Helicobacter pylori is considered as a diagnostic antigen. Therefore, this antigen can be used to detect H. pylori infection by stool immunoassays such as ELISA. The aim of this study was to simplify the AhpC protein purification procedures.
For whole cell protein extraction, the bacterial cells were ruptured by octly-β-D glucopyranoside. The isolation and purification of AhpC protein were attempted by various techniques including ammonium sulfate precipitation, dialysis, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution.
A simple method was used for protein purification AhpC protein. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high resolution capacity of this technique leads to a high level of purity of the protein. Moreover, it avoids contamination by other non-specific proteins which often appear during protein purification by column chromatography.
The present method is simple, rapid and makes it possible to preparate AhpC from H. pylori.
PMCID: PMC3468971  PMID: 23112994
Alkyl hydroperoxide reductase; AhpC; Electroelution; Helicobacter pylori; SDS-PAGE
18.  Analytical and preparative applications of magnetic split-flow thin fractionation on several ion-labeled red blood cells 
Magnetic Split-flow thin (SPLITT) fractionation is a newly developed technique for separating magnetically susceptible particles. Particles with different field-induced velocities can be separated into two fractions by adjusting applied magnetic forces and flow-rates at inlets and outlets.
Magnetic particles, Dynabeads, were used to test this new approach of field-induced velocity for susceptibility determination using magnetic SF at different magnetic field intensities. Reference measurements of magnetic susceptibility were made using a superconducting quantum interference device (SQUID) magnetometer. Various ion-labeled red blood cells (RBC) were used to study susceptibility determination and throughput parameters for analytical and preparative applications of magnetic SPLITT fractionation (SF), respectively. Throughputs were studied at different sample concentrations, magnetic field intensities, and channel flow-rates.
The susceptibilities of Dynabeads determined by SPLITT fractionation (SF) were consistent with those of reference measurement using a superconducting quantum interference device (SQUID) magnetometer. Determined susceptibilities of ion-labeled RBC were consistent within 9.6% variations at two magnetic intensities and different flow-rates. The determined susceptibilities differed by 10% from referenced measurements. The minimum difference in magnetic susceptibility required for complete separation was about 5.0 × 10-6 [cgs]. Sample recoveries were higher than 92%. The throughput of magnetic SF was approximately 1.8 g/h using our experimental setup.
Magnetic SF can provide simple and economical determination of particle susceptibility. This technique also has great potential for cell separation and related analysis. Continuous separations of ion-labeled RBC using magnetic SF were successful over 4 hours. The throughput was increased by 18 folds versus early study. Sample recoveries were 93.1 ± 1.8% in triplicate experiments.
PMCID: PMC1779266  PMID: 17177988
19.  GFP Facilitates Native Purification of Recombinant Perlucin Derivatives and Delays the Precipitation of Calcium Carbonate 
PLoS ONE  2012;7(10):e46653.
Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO3− as the first ionic interaction partner, but not necessarily for Ca2+. The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.
PMCID: PMC3463529  PMID: 23056388
20.  The Affinity Grid: A pre-fabricated EM grid for monolayer purification 
Journal of molecular biology  2008;382(2):423-433.
We have recently developed “monolayer purification” as a rapid and convenient technique to produce specimens of His-tagged proteins or macromolecular complexes for single particle electron microscopy (EM) without prior biochemical purification. Here, we introduce the “Affinity Grid”, a pre-fabricated EM grid featuring a dried lipid monolayer that contains Ni-NTA lipids (lipids functionalized with a Nickel-nitrilotriacetic acid group). The Affinity Grid, which can be stored for several months under ambient conditions, further simplifies and extends the use of monolayer purification. After characterizing the Affinity Grid, we used it to isolate, within minutes, ribosomal complexes from E. coli cell extracts containing His-tagged rpl3, the human homolog of the E. coli 50S subunit rplC. Depending on the way the sample was applied to the Affinity Grid, ribosomal complexes with or without associated mRNA could be prepared. Vitrified Affinity Grid specimens could be used to calculate three-dimensional reconstructions of the 50S ribosomal subunit as well as the 70S ribosome and 30S ribosomal subunit from images of the same sample. In addition, we established that Affinity Grids are stable for some time in the presence of glycerol and detergents. This feature allowed us to isolate His-tagged aquaporin-9 (AQP9) from detergent-solubilized membrane fractions of Sf9 insect cells. The Affinity Grid can thus be used to prepare single particle EM specimens of soluble complexes and membrane proteins.
PMCID: PMC2564605  PMID: 18655791
monolayer purification; lipid monolayer; affinity purification; single particle; cryo-electron microscopy
21.  Methods in Elastic Tissue Biology: Elastin Isolation and Purification 
Methods (San Diego, Calif.)  2008;45(1):32-41.
Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60–70 kDa monomer call tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin ‘contaminates’ are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein’s extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.
PMCID: PMC2405817  PMID: 18442703
elastin; elastic fiber; tropoelastin; microfibrils; fibrillin; purification
22.  Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography 
The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.
PMCID: PMC2925877  PMID: 20216526
23.  Biophysical characterization of G-protein coupled receptor-peptide ligand binding 
G-protein coupled receptors (GPCRs) are ubiquitous membrane proteins allowing intracellular response to extracellular factors that range from photons of light to small molecules to proteins. Despite extensive exploitation of GRCRs as therapeutic targets, biophysical characterization of GPCR-ligand interactions remains challenging. In this minireview, we focus on techniques which have been successfully employed for structural and biophysical characterization of peptide ligands binding to their cognate GPCRs. The techniques reviewed include solution-state nuclear magnetic resonance (NMR) spectroscopy; solid-state NMR; X-ray diffraction; fluorescence spectroscopy and single molecule fluorescence methods; flow cytometry; surface plasmon resonance; isothermal titration calorimetry; and, atomic force microscopy. The goal herein is to provide a cohesive starting point to allow selection of techniques appropriate to the elucidation of a given GPCR-peptide interaction.
PMCID: PMC3096653  PMID: 21455262 CAMSID: cams1714
G-protein coupled receptors; nuclear magnetic resonance spectroscopy; surface plasmon resonance; fluorescence spectroscopy; structural biology
24.  Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation 
Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices.
Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile.
Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge.
PMCID: PMC3163181  PMID: 21824404
25.  Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. 
Nucleic Acids Research  1998;26(22):5109-5115.
A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.
PMCID: PMC147948  PMID: 9801307

Results 1-25 (942989)