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PubMed2006

Application of a new vector system for efficient protein purification in the crystallization of PA5104/ORF from Pseudomonas aeruginosa.

We have developed a new T7-based vector system for rapid purification and high-throughput capability applicable for structural studies. The system allows purification of target proteins to homogeneity in two steps with a single Ni-affinity column. The first step relies on affinity purification of the N-terminal His-tagged protein in the conventional way, eluting the protein with imidazole. Addition of a His-tagged 3C protease to cleave the His-tag permits a second pass through the nickel column, this time all impurities bind to the column while the pure protein does not. This has the major advantage of quickly removing the residual contaminating proteins that are associated with nickel affinity purification as well as the protease and His-tag. Here, we describe the application of this system to over-express and purify ORF PA5104 from Pseudomonas aeruginosa. The protein was successfully crystallized and crystals were shown to diffract to atomic resolution. Additionally preliminary X-ray diffraction analysis of two crystals forms is presented, one diffracting to 1.9 A and the other to 0.96 A resolution.

Bacterial Proteins↗
PubMed1982

Purification by affinity chromatography of the glycine receptor of rat spinal cord.

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 +/- 6,000 was calculated for the glycine receptor protein.

Affinity Labels↗
PubMed1986

Use of monoclonal antibodies to separate the enantiomers of abscisic acid.

The resolution of racemates often requires difficult and time consuming purification procedures. McAb technology allows the production of specific antibodies in quantities suitable for the preparation of matrices for large scale affinity purification. Here we report the rapid separation of abscisic acid (ABA) enantiomers by affinity chromatography using McAb. This method appears to be far superior to previously published separations based on crystallization, chromatography, and affinity purification with conventional antisera. The approach here described will be particularly attractive in a wide variety of similar situations.

Abscisic Acid↗
PubMed1999

General expression vectors for production of hydrophobically tagged immunogens for direct iscom incorporation.

A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombinant immunogen as fused to a composite tag comprising one domain suitable for affinity purification and a hydrophobic tag designed for direct incorporation through hydrophobic interaction of the affinity-purified immunogen into an adjuvant system, in this case immunostimulating complexes (iscoms). Three different hydrophobic tags were evaluated: (i) a tag denoted IW containing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-dependent in such a way that an amphiphatic alpha-helix would be formed at low pH. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient M5-specific antibody responses upon immunization of mice, confirming successful incorporation into iscoms. The implications of these results for the design and production of subunit vaccines are discussed.

Amino Acid Sequence↗
PubMed1992

Affinity chromatographic purification of bovine lung endothelin receptor using biotinylated endothelin and avidin-agarose.

Endothelin receptor was purified from bovine lung by affinity chromatography using biotinylated endothelin and avidin-agarose. Endothelin was biotinylated with sulphosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate, a reactive form of biotin with a cleavable spacer arm containing a disulphide bond designed for a simple elution by thiols. Starting from 3.5 kg of bovine lung, about 200 micrograms of pure receptor were obtained.

Amino Acid Sequence↗
PubMed1989

Interaction of human lactoferrin with DNA: one-step purification by affinity chromatography on single-stranded DNA-agarose.

Human lactoferrin has been purified to apparent homogeneity with high recovery (greater than 95%) in one chromatographic step using immobilized single-stranded DNA-agarose. Human colostral whey, at neutral pH, was loaded onto columns of DNA-agarose; a single purified protein, lactoferrin, was eluted using elevated ionic strength buffers. The lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, and reverse-phase (C18) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels and silver staining showed the purified lactoferrin preparation to contain a single protein of 78 kD with intact immunologic determinants. Similar results were obtained before and after iron saturation of the colostral whey proteins. Apolactoferrin purified in this manner was shown to bind iron with high affinity. These results demonstrate the effectiveness of immobilized DNA as one of the most rapid and complete lactoferrin purification procedures described thus far.

Chromatography, Affinity↗
PubMed2005

Comparison of ELISA-based tyrosine kinase assays for screening EGFR inhibitors.

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu,Tyr)l:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431 cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.

Animals↗
PubMed1984

Affinity chromatographic purification and characterization of two iodinated tetanus toxin fractions exhibiting different binding properties.

Highly purified iodinated tetanus toxin preparations separate on ganglioside affinity columns into two distinct (A and B) fractions representing about 20% and 75% of the iodinated toxin, respectively. Fraction A, eluted by 1% NaCl, migrates like native tetanus toxin (150,000 mol. wt) on SDS polyacrylamide gel electrophoresis. It forms an aggregate of molecular weight approximately 360,000 on Sephacryl S-300 gel permeation chromatography in the presence of detergent and contains two isoforms on preparative chromatofocusing. Fraction A binds poorly to neurons in tissue culture or to synaptosomal membrane preparations. It retains, however, its antigenicity and biotoxicity. Fraction B, eluted by 6% NaCl, binds effectively to gangliosides and also to neurons or synaptosome preparations. It has a similar molecular weight and chain composition to the native toxin and displays two isoforms, precipitable during chromatofocusing. Fraction B possesses similar binding, immunological and toxic properties to the original iodinated tetanus toxin. Following excessive iodination (4-6 mCi/mg protein), toxicity can be remarkably reduced. Unlabeled toxin shows a similar chromatographic pattern to the iodinated toxin on affinity columns, suggesting that a large portion (30% by protein and 55% by toxicity) of the toxin has a poor affinity for gangliosides. The molecular pharmacokinetics of tetanus toxin with respect to affinity toward ganglioside-dependent and ganglioside-independent receptors needs re-evaluation.

Animals↗
PubMed1975

Affinity-chromatography purification of alkaline phosphatase from calf intestine.

A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.

Adsorption↗
PubMed2006

Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures.

Many mammalian proteins are multifunctional proteins with biological activities whose characterization often requires in vitro studies. However, these studies depend on generation of sufficient quantities of recombinant protein and many mammalian proteins cannot be easily expressed and purified as full-length products. One example is the Wilm's tumor gene product, WT1, which has proven difficult to express as a full-length purified recombinant protein using standard approaches. To facilitate expression of full-length WT1 we have developed approaches that optimized its expression and purification in Escherichia coli and mammalian cells. First, using a bicistronic vector system, we successfully expressed and purified WT1 containing a C-terminal tandem affinity tag in 293T cells. Second, using a specific strain of E. coli transformed with a modified GST vector, we successfully expressed and purified N-terminal GST tagged and C-terminal 2x FLAG tagged full-length human WT1. The benefits of these approaches include: (1) two-step affinity purification to allow high quality of protein purification, (2) large soluble tags that can be used for a first affinity purification step, but then conveniently removed with the highly site-specific TEV protease, and (3) the use of non-denaturing purification and elution conditions that are predicted to preserve native protein conformation and function.

Chromatography, Affinity↗
PubMed1975

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).

Chromatography, Affinity↗
PubMed1990

E. coli penicillin acylase: purification by affinity chromatography and covalent binding to nylon.

Penicillin acylase (EC 3.5.1.11) from E. coli, both in solution and immobilized on solid supports, has been commercially exploited for the large scale production of 6-aminopenicillanic acid (6-APA), which is an important intermediate for the manufacturing of semisynthetic penicillins. In this paper a very simple procedure of penicillin acylase purification is reported, which employs only one affinity chromatographic step (Sepharose-phenylacetic column). The enzyme was obtained at a high degree of purity and could be used for immobilization on partially hydrolyzed and activated nylon. Since the support is chemically inert and mechanically stable the catalyst can be used several times without any significant loss of activity, making the process of great commercial importance.

Chromatography, Affinity↗
PubMed1983

Affinity chromatographic purification of horse muscle acylphosphatase: evidence of the existence of multiple molecular forms.

Acylphosphatase was purified from horse muscle by a new procedure involving an affinity chromatography step and subsequent ion-exchange chromatography. This procedure was considerably milder than the preceding one, gave an overall yield of about 60% of activity and permitted isolation of three molecular forms with acylphosphatase activity. All these enzymatic forms are tightly bound to Sepharose 4B-linked anti-horse muscle acylphosphatase antibodies. Two of these forms (Ho1 and Ho3) are present in larger amounts: Ho1 corresponds to the enzyme purified according to the older procedure; this enzyme is a mixed disulfide between a main chain of 98 amino acid residues and glutathione. Ho2 differs from Ho1 only in the chemical nature of the molecule(s) S-S bound to the sole cysteine present at position 21 of the main chain. Ho3 is an S-S dimer of the main polypeptide chain. Ho1, Ho2, and Ho3 elicit very similar kinetic parameters in the presence of benzoylphosphate as a substrate.

Acid Anhydride Hydrolases↗