High-throughput functional affinity purification of mannose binding proteins from Oryza sativa

We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using alpha-D-mannose as the ligand. Bound fractions were eluted and subjected to one-dimensional electrophoresis, followed by high-performance liquid chromatography-tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue-specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high-throughput fashion.     

Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis

Protein ubiquitylation is a central regulatory mechanism that controls numerous processes in plants, including hormone signaling, developmental progression, responses to biotic and abiotic challenges, protein trafficking and chromatin structure. Despite data implicating thousands of plant proteins as targets, so far only a few have been conclusively shown to be ubiquitylated in planta . Here we describe a method to isolate ubiquitin–protein conjugates from Arabidopsis that exploits a stable transgenic line expressing a synthetic poly- UBQ gene encoding ubiquitin (Ub) monomers N-terminally tagged with hexahistidine. Following sequential enrichment by Ub-affinity and nickel chelate-affinity chromatography, the ubiquitylated proteins were trypsinized, separated by two-dimensional liquid chromatography, and analyzed by mass spectrometry. Our list of 54 non-redundant targets, expressed by as many as 90 possible isoforms, included those predicted by genetic studies to be ubiquitylated in plants (EIN3 and JAZ6) or shown to be ubiquitylated in other eukaryotes (ribosomal subunits, elongation factor 1α, histone H1, HSP70 and CDC48), as well as candidates whose control by the Ub/26S proteasome system is not yet appreciated. Ub attachment site(s) were resolved for a subset of these proteins, but surprisingly little sequence consensus was detected, implying that specific residues surrounding the modified lysine are not important determinants for ubiquitylation. We also identified six of the seven available lysine residues on Ub itself as Ub attachment sites, together with evidence for a branched mixed-linkage chain, suggesting that the topologies of Ub chains can be highly complex in plants. Taken together, our method provides a widely applicable strategy to define ubiquitylation in any tissue of intact plants exposed to a wide range of conditions.     

HaloTag-based purification of functional human kinases from mammalian cells

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.     

Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.     

Expression and affinity purification of recombinant proteins from plants

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system.     

Tandem affinity purification and identification of protein complex components

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.     

Tandem affinity purification of miRNA target mRNAs (TAP-Tar)

MicroRNAs (miRNAs) bind to Argonaute proteins, and together they form the RISC complex and regulate target mRNA translation and/or stability. Identification of mRNA targets is key to deciphering the physiological functions and mode of action of miRNAs. In mammals, miRNAs are generally poorly homologous to their target sequence, and target identification cannot be based solely on bioinformatics. Here, we describe a biochemical approach, based on tandem affinity purification, in which mRNA/miRNA complexes are sequentially pulled down, first via the Argonaute moiety and then via the miRNA. Our ‘TAP-Tar’ procedure allows the specific pull down of mRNA targets of miRNA. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.     

Tandem affinity purification vectors for use in gram positive bacteria

Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 degrees C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3xFLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product.     

Secretion and affinity purification of glutathione S-transferase fusion proteins from yeast

Summary Sequences encoding GST-fusion proteins were cloned into the Saccharomyces cerevisiae secretion vector, pYEX-S1, to direct secretion into the culture medium. GST and metallothionein fused to GST were secreted successfully and the fusion proteins purified. With several other GST-fusion proteins however, the proteins were retained inside the cell, indicating limitations to the types of proteins that can be secreted from yeast.     

One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1

The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis. However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature. Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria. The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L). The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.     

Expression and purification of functional human alpha-1-Antitrypsin from cultured plant cells

Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.     

Tandem affinity purification in Drosophila: the advantages of the GS-TAP system

Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material.     

The use of dye-ligand affinity chromatography for the purification of non-enzymatic human plasma proteins

Literature data are analysed in this review on the use of immobilized triazine dyes for the characterization, isolation and purification of non-enzymatic human plasma proteins in both conventional and high-pressure liquid chromatography systems. Attention is focused on the mode of interaction between the dyes and these proteins, as well as on the advantages over previously reported techniques. Future developments are discussed.     

Two-stage affinity purification for inducibly phosphorylated membrane proteins

Characterisation of tyrosine phosphorylations induced in immune cells in response to inflammatory stimuli may help elucidate the molecular bases of the diversity of immune responses. We have used anti-phosphotyrosine antibodies in combination with cell surface biotinylation in a two-step affinity purification procedure to recover pervanadate-induced tyrosine phosphorylated proteins from sub-cellular compartments, including the cell surface, of murine T cells and macrophages prior to separation by solution-phase isoelectric focussing and one-dimensional gel electrophoresis and identification by tandem mass spectrometry.     

Statistical optimization for immobilized metal affinity purification of secreted human erythropoietin from Drosophila S2 cells

We used a novel approach to affinity purify human erythropoietin (hEPO) following its secretion from Drosophila melanogaster S2 cells. Immobilized metal affinity purification of hEPO was optimized using a two-step serial statistical optimization strategy. After determining the elution conditions (based on preliminary batch-type purification experiments), the first optimization step considered three purification factors; resin, equilibrium, and washing. The results of this analysis showed that the resin amount was the major factor influencing yield and purity in both model equations and the washing factor lowered the confidence limits of the acquired model equations. The washing conditions were then set based on the results of the first step optimization and the second step then optimized three factors; resin, equilibrium, and elution. The yield and purity of hEPO were then compared following purification using three different approaches; batch-type purification based upon the conditions determined by serial statistical optimization, batch-type purification performed in preliminary experiments, and FPLC column chromatography-type purification. We found that the serial statistical optimization approach provided the best combination of yield and purity. These findings indicate that serial statistical optimization strategies can be successfully employed for immobilized metal affinity protein purification using either batch-type or column approaches.     

Peroxidase from cultured carrot cells: affinity purification and characteristics

Peroxidase, from cultured carrot cells, has been isolated and purified over Concanavalin-A-Sepharose by lectin affinity chromatography. The enzyme protein registers a gradual enhancement in activity concomitant with the growth of callus. Extracellular (ECP) & intracellular (ICP) peroxidases have been demonstrated in suspension cultures of carrot.     

Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma

Metal-chelating ligand and/or comonomer 2-methacrylolyamidohistidine (MAH) was synthesized by using methacryloyl chloride and L-histidine methyl ester. MAH was characterized by NMR and FTIR. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of methylmethacrylate (MMA) and MAH carried out in an aqueous dispersion medium. Poly(MMA-MAH) beads had a specific surface area of 37.5 m(2)/g. Poly(MMA-MAH) beads were characterized by water uptake studies, FTIR, SEM and elemental analysis. Elemental analysis of MAH for nitrogen was estimated as 34.7 microM/g of polymer. Then, Cu(2+) ions were chelated on the beads. Cu(2+)-chelated beads with a swelling ratio of 38% were used in the adsorption of human-immunoglobulin G (HIgG) from both aqueous solutions and human plasma. The maximum adsorption capacities of the Cu(2+)-chelated beads were found to be 12.2 mg/g at pH 6.5 in phosphate buffer and 15.7 mg/g at pH 7.0 in MOPS. Higher adsorption value was obtained from human plasma (up to 54.3 mg/g) with a purity of 90.7%. The metal-chelate affinity beads allowed one-step separation of HIgG from human plasma. The adsorption-desorption cycle was repeated 10 times using the same beads without noticeable loss in their HIgG adsorption capacity.