Purification of transforming growth factor-beta 1 binding proteins from porcine uterus membranes.

We have identified several transforming growth factor-beta 1 (TGF-beta 1) binding proteins in solubilized and glycoprotein-enriched porcine uterus membrane fractions by affinity cross-linking and in-gel ligand binding using 125I-labeled TGF-beta 1. By a ligand affinity chromatography using a column of immobilized recombinant TGF-beta 1, four components of apparent molecular weights 160,000, 80,000, 50,000, and 40,000 under reducing conditions were eluted at a pH of 3.5; the 160-,80-, and 40-kDa components were demonstrated to bind TGF-beta 1 specifically by the 125I-TGF-beta 1 binding assays. Further purification was performed by gel chromatography using a Superose 12 column eluted in 70% formic acid. The 40-kDa component was purified to an apparently homogenous form, whereas the 160-kDa component eluted in a broad peak overlapping the peak of the 80-kDa component. It remains to be elucidated whether these TGF-beta 1 binding proteins are related to cell surface receptors for TGF-beta s.     

Isolation of the vitellogenin-binding protein from locust ovaries

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-β-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.     

Evaluation of equilibrium gel filtration chromatography for the study of protein binding of aluminum in normal and uremic sera

Normal and uremic serum samples were chromatographed on a gel filtration column and fractions were analyzed for protein and aluminum content. Whereas much of the aluminum in the serum samples eluted with protein, a significant fraction appeared to be associated with low molecular weight species. Analysis of column fractions for aluminum binding by a competitive binding assay indicated no aluminum-binding serum constituents in these later eluting fractions. Uremic serum appeared, however, to contain aluminum-binding constituents of a lower molecular weight than in normal serum.     

Purification of tissue forms (Amastigotes) of Trypanosoma cruzi by immunoaffinity chromatography

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Sepharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

Purification of the c-fos enhancer-binding protein.

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Biosensor analysis of drug-target interactions: direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors

The sensitivity of BIACORE technology is sufficient for detection and characterization of binding events involving low-molecular-weight compounds and their immobilized protein targets. The technology requires no labeling and provides information on the stability of the compound/target complex with a single injection of the compound. This is useful for qualifying hits obtained in a primary screen and in lead optimization. Although immobilized targets can be reused, the surface may slowly deteriorate, solvent effects can distort binding levels during injection of compounds, and some compounds may exhibit broad protein selectivity rather than target specificity. A reliable direct binding assay for compounds binding to immobilized thrombin using a combination of two reference surfaces, a dextran surface for subtraction and calibration of solvent effects and a protein surface for identification of compounds that tend to bind proteins, has been developed. Eleven compounds with known binding specificity to thrombin and 159 additional compounds were investigated. All compounds with known binding specificity were identified at 1 and 10 microM concentration. One additional compound was scored as positive. The direct binding assay compared favorably with two competitive assay formats, a surface competitive assay and a inhibitor in solution assay, that were examined in parallel.     

Purification of hamster dihydroorotate synthetase using Procion blue-Sepharose

Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de novo pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-Sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 mumol/min/mg protein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure.     

Solubilization and partial purification of GABAB receptor from bovine brain

gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.     

Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex

We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclin and development of ELISA for anti-PCNA antibodies

Proliferating cell nuclear antigen (PCNA), also called cyclin, was purified from PBS extract of rabbit thymus by using a combination of ammonium sulfate fractionation, DEAE-Sephacel, HPLC ion exchange, and HPLC gel filtration column chromatography. PCNA was purified more than 600 times and was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE showed that a 36 kD protein was selectively isolated in this purification process, and this protein was identified as PCNA by immunoblotting. Other previously identified nuclear antigens, Sm, nRNP, SS-A/Ro, SS-B/La, histone, and DNA, were not detected in this preparation by counterimmunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). Purified PCNA was used as an antigen to develop ELISA for rapid and specific detection of anti-PCNA in human sera. For further purification, the 36 kD band was electrophoretically eluted from SDS gel slices. The amino acid composition and the first 25 residues from the N-terminus of the protein were determined by using electroeluted PCNA. This amino acid sequence was found to be unique and showed little sequence homology with existent proteins in the protein identification resources databank.     

Affinity chromatography of protein kinase C-phorbol ester receptor on polyacrylamide-immobilized phosphatidylserine

An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.     

Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain

N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.     

Artifact-inducing enrichment of ethylenediaminetetraacetic acid and ethyleneglycoltetraacetic acid on anion exchange resins

Multivalent metal chelators, ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA), are used extensively during protein purification. Both strong (Q) and weak (DEAE) anion exchange resins were found to adsorb surprisingly large quantities of EDTA and EGTA that elute from the resin at NaCl concentrations of approximately 240 mM (EDTA) and 140 mM (EGTA). The EDTA/EGTA elution and saturation parameters were determined for five commonly used anion exchange resins. The resulting concentration of eluted EDTA was 10- to 200-fold higher than that originally present in the sample or in the mobile phase. Samples from fractions containing such a high concentration of EDTA were found to inhibit Mg²⁺-dependent polymerase chain reaction (PCR). EDTA binding to the anion exchange resins could saturate the resin, decrease its binding capacity, and displace weakly bound proteins such as green fluorescent protein (GFP). Several steps are suggested to minimize on-column EDTA concentration, including column equilibration in the absence of any EDTA, lower concentrations (0.1–0.5 mM) of EDTA, monitoring eluate absorbance at 280 nm as well as at 215 nm, adding EDTA back into fractions eluting before the EDTA peak, and performing blank column runs to control for the effect of changes in EDTA concentration in downstream assays.     

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.