Dinucleotide cap analogue affinity resins for purification of proteins that specifically recognize the 5' end of mRNA

Here we present first dinucleotide affinity resins for purification of proteins that specifically recognize the 5' end of mRNA. Constructed resins possess either a naturally occurring mono- or trimethylated cap or their analogues resistant towards enzymatic degradation, bearing a CH(2) bridge between β and γ position of the 5',5'-triphosphate chain. All cap analogues were attached to a polymer support (EAH-Sepharose) through the carboxylic group that had been generated by derivatization of the 2',3'-cis diol of the second nucleotide in the cap structure with levulinic acid.     

A unified method for purification of basic proteins

Protein purification is still very empirical, and a unified method for purifying proteins without an affinity tag is not available yet. In the postgenomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied for purifying any recombinant basic protein from Escherichia coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI−1 ? 6.94) may be purified from E. coli in a single step using a cation-exchanger resin, SP-Sepharose, and a selected buffer pH, depending on the pI of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step.     

A method for determining binding kinetics applied to thiamine-binding protein

A rapid and simple method for assaying the binding activity of thiamine-binding protein is described. By this assay method, the binding characteristics of rice bran thiamine-binding protein have been evaluated with [14C]thiamine as ligand. Analysis of these data by Scatchard plot resulted in linear plots giving a dissociation constant (Kd) for thiamine of 0.55 microM and a maximum binding (Bmax) of 14.5 pmol of ligand bound/microgram of protein. Thiamine binding to the binding protein was time dependent and reached equilibrium at approximately 20 min. The Kob was 0.18 min-1 and the k1 was 1.25 X 10(5) min-1 M-1. Reversibility of thiamine binding at equilibrium was completed at 60 min with a k2 value of 0.052 min-1. The Kd calculated from the reverse rate constant was 0.42 microM. These results indicated that this binding assay method was substantially reliable and accurate.     

Purify First: rapid expression and purification of proteins from XMRV

Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale.     

A general platform for antibody purification utilizing engineered-micelles

We introduce a new concept and potentially general platform for antibody (Ab) purification that does not rely on chromatography or specific ligands (e.g., Protein A); rather, it makes use of detergent aggregates capable of efficiently capturing Ab while rejecting hydrophilic impurities. Captured Ab are then extracted from the aggregates in pure form without co-extraction of hydrophobic impurities or aggregate dissolution. The aggregates studied consist of conjugated “Engineered-micelles” built from the nonionic detergent, Tween-20; bathophenanthroline, a hydrophobic metal chelator, and Fe²⁺ions. When tested in serum-free media with or without bovine serum albumin as additive, human or mouse IgGs were recovered with good overall yields (70–80%, by densitometry). Extraction of IgGs with 7 different buffers at pH 3.8 sheds light on possible interactions between captured Ab and their surrounding detergent matrix that lead to purity very similar to that obtained via Protein A or Protein G resins. Extracted Ab preserve their secondary structure, specificity and monomeric character as determined by circular dichroism, enzyme-linked immunosorbent assay and dynamic light scattering, respectively.     

The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins

The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. Mutagenesis techniques have been widely used to modulate apolipoprotein lipid affinity for studying biological function, but the approach requires rapid and reliable lipid affinity assays to compare the mutants. Here, we describe a novel method that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUVs) containing trace biotinylated and fluorescent phospholipids. After a 30 min incubation at various apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted and measured in a fluorescence microplate reader for calculation of a dissociation constant and the maximum number of potential binding sites on the SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV, and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations. The assay is easy to perform and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 h, consuming only 120 microg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.     

A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents

Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein–protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10⁴ pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner.     

Cloning, expression and purification of the CCN family of proteins in Escherichia coli

The CCN proteins are extracellular matrix associated proteins involved in critical cell activities and several aggressive forms of cancer. The proteins share a modular structure of four discrete domains and 38 conserved cysteine residues. The absence of any structural information of these proteins has resulted in a need for the ability to produce substantial amounts of pure CCN protein. Through bacterial expression and inclusion body based purification, pure recombinant CCN proteins have been produced for use in structural and biochemical experiments.     

Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials

A range of studies were carried out to investigate the underlying reason for differences in dynamic binding capacities observed with various antibodies and Fc-fusion proteins during Protein A chromatography. Dynamic binding capacities were determined for these biomolecules using different protein A stationary phase materials. SEC was carried out to determine the relative sizes of the antibodies and fusion proteins. Pore diffusivities and static binding capacities were also determined on these Protein A resin materials. Trends in the dynamic binding capacities for these molecules did not correlate with differences in pore diffusion coefficients as might be expected for a mass transfer limited system. Instead, dynamic binding capacities were seen to follow the same trends as the static binding capacities and the apparent size of the molecules. Differences in static binding capacities were attributed to be due to differences in steric factor between the molecules. Solution binding stoichiometry studies were employed to estimate intra-Protein A steric effects while binding to the various domains within a Protein A ligand. In addition, steric hindrance was also found to exist between adjacent immobilized Protein A ligands on the chromatographic surface. The combination of intra and inter Protein A steric hindrances can explain differences in binding capacities observed between various antibody and Fc fusion proteins. The effect of Protein A ligand density on these supports was also examined and the results indicate that increasing Protein A ligand density leads to a situation of diminishing returns for binding capacity due to increased steric hindrance on the resin surface. The results presented in this paper show that steric hindrances can dominate over mass transfer effects in causing capacity variation between different molecules on the same stationary phase. This can lead to the development of more cost-efficient chromatographic stationary phases as well as provide information during the selection of Protein A media for preparative purification of monoclonal antibodies and Fc fusion proteins.     

Application of inline variable pathlength technology for rapid determination of dynamic binding capacity in downstream process development of biopharmaceuticals

Determination of dynamic binding capacity (DBC) for capture purification chromatographic step is usually the first experiment to be performed during downstream process development of biopharmaceuticals. In this work, we investigated the application of inline variable pathlength technology using FlowVPE for rapid determination of DBC on affinity resins for protein capture and proved its comparability with offline titer methods. This work also demonstrated that variable pathlength technology for DBC determination can be successfully applied to different classes of monoclonal antibodies and fusion proteins. This enabled rapid screening of affinity resins and optimization of the capture chromatography step. Hence, use of inline variable pathlength technology eliminated the dependency on offline titer data, traditionally used for DBC determination and accelerated overall process development timelines with less cost.     

A standardized method for determining buffering capacity of plant foliage

Summary A method for determining the buffering capacity (B.C.) of foliage extracts was standardized and evaluated. Sources of variations (biological, field and laboratory) were identified. These variations were reflected in inter-specific differences, seasonal fluctuations, age of the foliage and duration and the conditions of storage of the extracts. Procedures have been recommended to eliminate or minimize sources of variations (other than inherent specific) by standardizing the field sampling, laboratory processing and methods, and calculations of the buffering capacity. Plants such as lichens, known to be sensitive to air pollutants, had very low B.C. whereas species of intermediate sensitivity such as balsam fir had higher B.C. The B.C. being inherited and significantly different among species, has potential for its use in indexing the relative sensitivity of species to air pollutants especially in areas where large numbers of species are to be compared.     

A rapid and efficient method for purification of recombinant adenovirus with arginine-glycine-aspartic acid-modified fibers

Recombinant adenoviral vectors (adenovectors) have been subject to various genetic modifications to improve their transduction efficiency and targeting capacity. Production and purification of adenovectors with modified capsid proteins can be problematic using conventional two-cycle CsCl gradient ultracentrifugation. We have developed a new method for purifying recombinant adenovectors in two steps: iodixanol discontinuous density gradient ultracentrifugation and size exclusion column chromatography. The purity and infectious activity of adenovectors isolated by the two methods were comparable. The new method yielded three to four times more adenovectors with arginine-glycine-aspartic acid (RGD)-modified fiber proteins than did the conventional CsCl method. For other fiber-modified and wild-type adenovectors, the yields of the two methods were comparable. Thus, the iodixanol-based method can be used not only to improve the production of RGD-modified adenovectors but also to purify adenovectors with or without fiber modifications. Moreover, the whole procedure can be completed in 3h. Therefore, this method is rapid and efficient for production of recombination adenovectors, especially those with RGD-modified fibers.     

A modified method for determining protein binding capacity of plant polyphenolics using radiolabelled protein

A modified radiochemical protein binding method for determining the protein binding capacity of plant polyphenolics (tannins) is described. Purified tannin or unfractionated plant extracts were immobilised on filter paper discs and incubated with the 125I-labelled bovine serum albumin. Protein bound to the disc was proportional to the amount of tannin applied to the disc, although at high concentrations of polyphenolics the discs became saturated and the relationship was no longer applicable. The method was validated using purified procyanidin from Sorghum grain and has been applied to crude polyphenolic extracts from maple, white oak, black oak, walnut and tulip poplar leaves. Specific chemical assays for the determination of proanthocyanidins (acid butanol method) and hydrolysable tannins (modified potassium iodate method) were employed to validate the new protein binding method with the complex plant extracts.     

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.     

A simple and rapid method to measure cholesterol binding to P450s and other proteins

Cholesterol plays an important role in cellular function and membrane compartmentalization and is involved in the interaction with more than a dozen of different proteins. Using three cholesterol-metabolizing cytochrome P450s (P450s 7A1, 46A1, and 11A1), we have developed a rapid and simple assay for measurements of nanomolar to micromolar cholesterol affinities. In this assay, the P450 is incubated with a fixed amount of radiolabeled cholesterol and varying concentrations of cold cholesterol followed by separation of free and protein-bound cholesterol via filtration through a membrane. Free cholesterol is found in the flow-through fraction, whereas P450 binds to the membrane. The radioactivity of the membranes is then measured, and a saturation curve is generated after correction for nonspecific binding of cholesterol to the filter. The validity of the filter assay was confirmed by spectral assay, a traditional method to evaluate the interaction of the P450 enzymes with their substrates. Two types of membranes, one binding positively charged proteins and another binding negatively charged proteins, were identified. These membranes were also found to hold proteins through hydrophobic interactions. Thus, the cholesterol binding properties of a wide variety of proteins could be characterized using this filter assay.     

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.     

A simple and rapid method for the purification of the mitochondrial porin from mammalian tissues

A new, simple and rapid procedure for the purification of high amounts of mitochondrial porins from different tissues of mammalia is described. The method consists in a single step hydroxyapatite / celite chromatography of Triton X-100 solubilized mitochondrial membranes. For optimal purification several factors are critical such as the absence of salts, a low protein / detergent ratio and an exact hydroxyapatite / celite ratio of 2:1.     

A novel method for rapid production and purification of exfoliative toxin A of Staphylococcus aureus

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (UT516, UT519, ATCC 27957) were used to determine if bovine lactoferrin (Lf) binds to bacterial cells by biotin avidin binding assay (BABA), enzyme-linked immunosorbent assay (ELISA), and binding inhibition assay. Binding assays revealed that all strains of S. dysgalactiae subsp. dysgalactiae (S. dysgalactiae) evaluated in this study bound to Lf. However, differences in Lf binding capability among strains and between methods used were detected. Binding of Lf was not inhibited by transferrin (Tf) and Lf moiety molecules (mannose, galactose, and lactose) but by Lf. This study demonstrates that S. dysgalactiae bound to bovine Lf in a specific manner.     

A novel method for the purification of recombinant subunit I of theDolichos biflorus seed lectin

Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of theirin situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous over-expression. The larger subunit of theDolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090±16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.