Avidin Column Research Articles

A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events.

Whole Surface Image of Mycoplasma mobile, Suggested by Protein Identification and Immunofluorescence Microscopy

Mycoplasma mobile, a freshwater fish pathogen featured with robust gliding motility, binds to the surface of the gill, where it then colonizes. Here, to obtain a whole image of its cell surface, we identified the proteins exposed on the surface using the following methods. (i) The cell surface was labeled with sulfosuccinimidyl-6-(biotinamido) hexanoate and recovered by an avidin column. (ii) The cells were subjected to phase partitioning using Triton X-114, and the hydrophobic proteins were recovered. (iii) The membrane fraction was analyzed by two-dimensional gel electrophoresis. These recovered proteins were subjected to peptide mass fingerprinting, and a final list of 36 expressed surface proteins was established. The ratio of identified proteins to whole surface proteins was estimated through two-dimensional gel electrophoresis of the membrane fraction. The localization of three newly found proteins, Mvsps C, E, and F, has been clarified by immunofluorescence microscopy. Integrating all information, a whole image of the cell surface showed that the proteins for gliding that were localized at the base of the protrusion of flask-shaped M. mobile account for more than 12% of all surface proteins and that Mvsps, surface variants that were localized at both parts other than the neck, account for 49% of all surface proteins.

Acetyl-CoA Carboxylase Regulates Global Histone Acetylation

Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA in the nucleocytosolic compartment. However, because nucleocytosolic acetyl-CoA is also used for de novo synthesis of fatty acids, histone acetylation and synthesis of fatty acids compete for the same acetyl-CoA pool. The first and rate-limiting reaction in de novo synthesis of fatty acids is carboxylation of acetyl-CoA to form malonyl-CoA, catalyzed by acetyl-CoA carboxylase. In yeast Saccharomyces cerevisiae, acetyl-CoA carboxylase is encoded by the ACC1 gene. In this study, we show that attenuated expression of ACC1 results in increased acetylation of bulk histones, globally increased acetylation of chromatin histones, and altered transcriptional regulation. Together, our data indicate that Acc1p activity regulates the availability of acetyl-CoA for histone acetyltransferases, thus representing a link between intermediary metabolism and epigenetic mechanisms of transcriptional regulation.

Purification of untagged HIV-1 reverse transcriptase by affinity chromatography

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2 mM Mn 2+. The RT retained in the column was eluted after soaking overnight in 10 mM EDTA to retrieve the Mn 2+. In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.

Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

Thematic Review Series: Proteomics. Proteomic analysis of lipid-protein complexes

There is intense interest in comprehensive proteomic approaches for analyzing integral membrane proteins and lipoproteins. Key features of mass spectrometric analysis center on enriching biological material for proteins of interest, efficiently digesting them, extracting the resulting peptides, and using fractionation methods to comprehensively sample proteins or peptides by tandem mass spectrometry. However, lipid-associated proteins are generally rich in hydrophobic domains and are often low in abundance. These features, together with the associated lipid, make their mass spectrometric analysis technically challenging. In this article, we review analytical strategies for successful proteomic analysis of lipid-associated proteins.

Chemical labeling and enrichment of nitrotyrosine-containing peptides

Protein tyrosine nitration (PTN) is a post-translational modification of proteins associated with a number of inflammatory diseases. While PTN is rather selective (not all proteins are modified and within a protein, only certain tyrosines are subject to nitration), no consensus sequence has been identified. Since PTN is a low-abundance post-translational modification, it is necessary to enrich modified proteins and/or to detect them with high selectivity and sensitivity. Until now this has been mostly accomplished with anti-nitrotyrosine antibodies in combination with two-dimensional gel electrophoresis and mass spectrometry. We propose a chemical labeling approach designed to allow enrichment of tyrosine-nitrated peptides independent of the sequence context, which is a potential shortcoming of antibody-based approaches. In this procedure, all amines are blocked by acetylation followed by conversion of nitrotyrosine to aminotyrosine and biotinylation of aminotyrosine. The entire reaction sequence is performed in a single buffer with no need for sample cleanup or pH changes thereby reducing sample loss. Free biotin is subsequently removed with a strong cation exchanger, the labeled peptides are enriched on an immobilized avidin column and the enriched peptides analyzed by LC–MS/MS. As a proof of concept, this method was successfully applied to the enrichment of tyrosine-nitrated angiotensin II in a tryptic digest of bovine serum albumin (BSA). The approach presented here is well adapted to peptide analysis, for instance in shotgun proteomics.

Identification of New Amine Acceptor Protein Substrate Candidates of Transglutaminase in Rat Liver Extract: Use of 5-(Biotinamido) Pentylamine as a Probe

Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.

Integrated Membrane Protein Analysis of Mature and Embryonic Stem Cell-derived Smooth Muscle Cells Using a Novel Combination of CyDye/Biotin Labeling

Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between CyDye-tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated, and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha6, and CD98 (4F2) to be significantly up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.

Improved Tumor Targeting and Decreased Normal Tissue Accumulation through Extracorporeal Affinity Adsorption in a Two-Step Pretargeting Strategy

Evaluation of the possibilities of reducing the accumulation of radiolabeled streptavidin in radiosensitive organs by extracorporeal affinity adsorption (ECAT). Rats were injected with biotinylated antibody and subjected to removal of the antibodies from the circulation by ECAT 24 h after injection (avidin column). Animals were then injected with 111In-1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic acid (DOTA)-streptavidin. In a third step, animals were subjected to a second ECAT 8 h after injection to remove the DOTA-streptavidin from the circulation (biotin column). Biodistribution and tumor targeting of DOTA-streptavidin 24 h after injection was determined. Elimination of biotinylated antibody by ECAT before injection of DOTA-streptavidin increased the tumor targeting by 50%. In addition, the levels of DOTA-streptavidin in liver and lymph nodes were reduced by 60%, which implied a 4.3- and 3.8-fold increase of tumor-to-liver and tumor-to-lymph node ratios, respectively. By doing a second ECAT to remove DOTA-streptavidin from the circulation, accumulation in normal tissues was reduced. However, this latter ECAT also reduced tumor accumulation by 25% (mostly corresponding to radioactivity in the circulation). ECAT was efficient as a means of removing biotinylated antibodies and would probably also be efficient for the clearance of streptavidin-conjugated antibodies. Conversely, the use of ECAT for removal of radiolabeled streptavidin seems not to offer any advantage.

Isotope-Coded Affinity Tagging of Proteins

INTRODUCTIONQuantitative proteomics has traditionally been performed using 2D gel electrophoresis, where quantitation is accomplished by recreating differences in the staining patterns of proteins derived from two states of cell populations or tissues from a similar biological system. More recently, mass spectrometry (MS) methods based on stable isotope quantitation have been developed that show significant potential for differential expression proteomic studies. One such in vitro method, described in this protocol, involves the use of isotope-coded affinity tags (ICATs) with three functional moieties: a cysteine reactive moiety, a linker with either eight hydrogens (the light form of the reagent) or eight deuteriums (the heavy form of the reagent, having an isotope code or mass tag of 8 Da), and a biotin moiety (the affinity tag). Using this technique, the cysteine side chains in complex mixtures of proteins from two different states of a cell population (e.g., normal vs. disease) are reduced and alkylated using the light form of the reagent (d0-labeled tag) in one cell state and the heavy form of the reagent (d8-labeled tag) for proteins in the second cell state. The two mixtures (d0 and d8 labeled) are then combined and subjected to proteolytic digestion (typically, with trypsin and/or Lys-C). Generated cysteine-containing peptides are affinity-purified using an avidin column, resulting in a "simplified" mixture of peptides that contains ~10-fold fewer peptides than the original mixture. These peptides are analyzed by MS, and quantitation information based on the relative abundance of the d0 and d8 isotopes is obtained. The identification of proteins is obtained from the peptide molecular mass and MS/MS-derived amino acid sequence.

Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography‐stable isotope labeling and tandem mass spectrometry

We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.

Comparative proteomic approaches for the isolation of proteins interacting with thioredoxin

Thioredoxin (TRX) is a small multifunctional protein with a disulfide active site involved in redox regulation. To gain insight into the numerous proteins able to interact with thioredoxin in Arabidopsis thaliana, we have compared three different proteomic procedures. In the two first approaches targets present in a mixture of soluble leaf proteins were reduced by the cytosolic TRX h3, then the new thiols were labeled either with radioactive iodoacetamide allowing specific detection (first method) or with a biotinylated thiol-specific compound allowing selective retention on an avidin column (second method). The third method involved a chromatography on a mutated TRX h3 column, which is able to covalently trap potential targets. All together, the three approaches enabled us to propose 73 proteins as being TRX-linked, and involved in various processes. Methods 1 and 3 were not only efficient with respectively 47 and 41 potential targets, but also complementary as only 26% of the targets were identified by both procedures. The second method with only 12 proteins was less efficient. However, this approach, as well as the first one when coupled with differential labeling of the cysteine residues, could be more informative about the cysteines involved in the thiol-disulfide interchange.

CKAP4/p63 Is a Receptor for the Frizzled-8 Protein-related Antiproliferative Factor from Interstitial Cystitis Patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

“SMART” Drug Delivery Systems: Double-Targeted pH-Responsive Pharmaceutical Nanocarriers

To develop targeted pharmaceutical carriers additionally capable of responding to certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, biotin and TATp functions on the surface of nanocarriers were "shielded" by long protecting PEG chains (pH-degradable PEG(2000)-PE or PEG(5000)-PE) or by even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG(3400)-PE or PEG(5000)-PE). At pH 7.4-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-30 min) at lower pH values (pH 5.0-6.0), nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-Hz-PE and acquired the ability to become strongly retained on an avidin column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.

Towards NMR structural studies on the peripheral cannabinoid receptor CB2 in micellar solution.

Human peripheral-type cannabinoid receptor (CB2) is a G protein-coupled membrane receptor (GPCR) involved in the immune and hormonal response. Because of its importance in signal transduction pathways, this membrane protein has become a target for drug design. Development of novel ligands would benefit greatly from NMR structural studies on ligands in the receptor-bound state and the protein. Functional CB2 was successfully expressed in E.coli as a fusion with maltose-binding protein (MBP), thioredoxin (TrxA) and a (His)10 tag (construct 107). The receptor was purified by Ni-NTA affinity chromatography to about 80% purity as judged by silver staining of SDS-PAGE gels. In order to further purify the protein, several constructs were designed which carry a second tag. The construct 109, which carries a biotin ligase recognition site (BRS) downstream of the CB2 sequence, produced in vivo biotinylated protein. However, introduction of this tag did not significantly increase the purity of the protein after elution from a monomeric avidin column. Insertion of the BRS upstream of the CB2 sequence (construct 112) led to a low level of in vivo biotinylation. This protein was successfully biotinylated in vitro and cleaved with Tev protease. Purification of the protein with a monomeric avidin column resulted in a highly enriched CB2 fraction. The efficiency of Tev protease cleavage was tested on a series of CB2-fusions with one or two cleavage sites. Full cleavage of the fusion at mg-quantities was achieved in micellar solution at 4oC. The cleaved CB2-fusion in bacterial membranes retained functionality as shown by ligand binding. Construct 115, with a second decahistidine tag at the N-terminus of CB2, enabled efficient purification of the cleaved CB2 on a Ni-NTA column.

Identification of Leukemia Surface Proteins Using a Proteomic Technique

Background: Numerous cell surface proteins of leukemia cells such as CD33 and CD52 have been identified as diagnostic and therapeutic targets. Thus the profiling of the cell surface proteome and proteins restricted to specific leukemia(s) can provide a way to identify novel targets for leukemia diagnosis and therapy. However, there is a lack of data pertaining to the comprehensive analysis of surface membrane proteins because there are few effective strategies for profiling surface membrane proteomes. Methods: We report on the application of quantitative proteomic techniques that incorporate affinity- capture and purification on monomeric avidin columns to identify all biotinylated cell surface proteins from leukemia cell lines. Results: An analysis of a subset of biotinylated proteins among the different human leukemia cell lines using matrix-assisted laser desorption ionization and tandem mass spectrometry identified, among others, some widely expressed proteins in leukemia cells, such as CD11a, CD11c, CD18, CD31, CD44, and CD147, as well as a set of proteins identified as chaperone proteins, including HSP90, GRP78, GRP75, HSP70, HSP60 and protein disulfide isomerases. On the basis of their known functional roles, several of these proteins may participate in the progression of leukemogenesis and should be considered as potential markers of leukemia. Conclusion: Comprehensive profiling of the leukemia cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns to a specific cell line.

Brain PP2A is modified by thiol-disulfide exchange and intermolecular disulfide formation

The regulation of protein phosphatase 2A (PP2A) activity by thiol-disulfide exchange and resulting formation of an intermolecular disulfide was examined following exposure of a rat brain soluble fraction to a biotinylated derivative of the model disulfide HPDP (HPDP–biotin) which would be expected to label reactive protein thiols with a disulfide-linked biotin. The results show that a low concentration (500 μM) of HPDP–biotin produced substantial inhibition of PP2A activity and promoted the binding of the catalytic subunit of PP2A to an immobilized avidin-affinity column. Both the inhibition of PP2A activity and the binding of PP2A to the avidin column were reversed by treatment with the disulfide reducing agent dithiothreitol (DTT). Furthermore, the specific activity of PP2A was up to 7-fold higher in the DTT-eluted fractions from the avidin-affinity column than in the soluble fraction. These findings demonstrate directly that PP2A is susceptible to reversible inhibitory modification by thiol-disulfide exchange and provide mechanistic support for the emerging view that PP2A is an oxidant-sensitive protein phosphatase.

Solid-phase biotinylation of antibodies.

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.

Biotin–ubiquitin tagging of mammalian proteins in Escherichia coli

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin–ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin–ubiquitin tag from biotin–ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.