Metal Chelate Affinity Chromatography Research Articles

Purification of horse radish peroxidase by metal affinity chromatography in an expanded bed system

Immobilised metal chelate affinity chromatography (IMAC) in an expanded bed mode was used for the purification of horse radish peroxidase. Recovery of horse radish peroxidase varied between 85 and 72% starting from the crude homogenate. When a pure peroxidase was passed through the purification protocol a recovery of about 95% was achieved.

Production of the Islet Cell Antigen ICA69 (p69) with: Baculovirus Expression System: Analysis with a: Solid-phase Time-resolved Fluorescence Method of Sera: from Patients with IDDM and Rheumatoid Arthritis

Islet cell antigen 69 (ICA69), previously implicated as an autoantigen in autoimmune insulin-dependent diabetes mellitus (IDDM), was produced using baculovirus-mediated expression in Spodoptera frugiperda (Sf9) insect cells. In these cells the protein was effectively expressed and ICA69 carrying C-terminal histidine-hexapeptide could be efficiently purified using immobilized metal chelate affinity chromatography. Screening of patient and control sera using this protein as an antigen in time-resolved fluoroimmunoassay (TR-FIA) identified 4/50 of patients with IDDM and 6/73 of patients with rheumatoid arthritis (RA) to be positive for ICA69 antibodies. The number of positives did not differ significantly between patients and control subjects but the level of binding was higher in sera from RA patients compared to that of control sera (P = 0.003). The results show that some subjects have specific autoreactive antibodies against the ICA69 protein produced with recombinant technology.

The yiaE gene, located at 80.1 minutes on the Escherichia coli chromosome, encodes a 2-ketoaldonate reductase.

An open reading frame located in the bisC-cspA intergenic region, or at 80.1 min on the Escherichia coli chromosome, encodes a hypothetical 2-hydroxyacid dehydrogenase, which was identified as a result of the E. coli Genome Sequencing Project. We report here that the product of the gene (yiaE) is a 2-ketoaldonate reductase (2KR). The gene was cloned and expressed with a C-terminal His tag in E. coli, and the protein was purified by metal-chelate affinity chromatography. The determination of the NH2-terminal amino acid sequence of the protein defined the translational start site of this gene. The enzyme was found to be a 2KR catalyzing the reduction of 2, 5-diketo-D-gluconate to 5-keto-D-gluconate, 2-keto-D-gluconate (2KDG) to D-gluconate, 2-keto-L-gulonate to L-idonate. The reductase was optimally active at pH 7.5, with NADPH as a preferred electron donor. The deduced amino acid sequence showed 69.4% identity with that of 2KR from Erwinia herbicola. Disruption of this gene on the chromosome resulted in the loss of 2KR activity in E. coli. E. coli W3110 was found to grow on 2KDG, whereas the mutant deficient in 2KR activity was unable to grow on 2KDG as the carbon source, suggesting that 2KR is responsible for the catabolism of 2KDG in E. coli and the diminishment of produced 2KDG from D-gluconate in the cultivation of E. coli harboring a cloned gluconate dehydrogenase gene.

Retention of neutralising activity by recombinant anti-pneumolysin antibody fragments

The variable domains of a neutralising (prevents erythrocyte lysis) anti-pneumolysin monoclonal antibody have been cloned and expressed as functional protein in Escherichia coli. Purification of the anti-pneumolysin single-chain antibody fragment, via antibody-affinity or metal-chelate affinity chromatography, resulted in product that was predominantly in a dimeric or monomeric form, respectively. The dimeric single-chain antibody fragment showed a higher sensitivity and affinity for immobilised antigen in both ELISA and BIAcore studies. The dimeric single-chain antibody fragment was as effective at protecting erythrocytes from lysis as the parent monoclonal. The monomeric, low affinity single-chain antibody fragment, showed reduced neutralising potency. As antibiotic resistant Streptococcus pneumoniae strains continue to show an increasing word-wide distribution, recombinant, neutralising antibody fragments, may provide an additional class of molecules useful in the treatment of toxaemia.

Retention of neutralising activity by recombinant anti-pneumolysin antibody fragments.

The variable domains of a neutralising (prevents erythrocyte lysis) anti-pneumolysin monoclonal antibody have been cloned and expressed as functional protein in Escherichia coli. Purification of the anti-pneumolysin single-chain antibody fragment, via antibody-affinity or metal-chelate affinity chromatography, resulted in product that was predominantly in a dimeric or monomeric form, respectively. The dimeric single-chain antibody fragment showed a higher sensitivity and affinity for immobilised antigen in both ELISA and BIAcore studies. The dimeric single-chain antibody fragment was as effective at protecting erythrocytes from lysis as the parent monoclonal. The monomeric, low affinity single-chain antibody fragment, showed reduced neutralising potency. As antibiotic resistant Streptococcus pneumoniae strains continue to show an increasing word-wide distribution, recombinant, neutralising antibody fragments, may provide an additional class of molecules useful in the treatment of toxaemia.

Encephalitogenicity of myelin-associated oligodendrocytic basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase for BALB/c and SJL mice.

In search of new encephalitogenic myelin antigens, the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and 19 000 MW isoform of myelin-associated oligodendrocytic basic protein (MOBP) were obtained as recombinant proteins by the baculovirus expression system in Spodoptera frugiperda cells and purified to homogeneity by immobilized metal chelate affinity chromatography (IMAC). The purified MOBP was soluble in water and showed retarded migration on sodium dodecyl sulphate-polyacrylamide gel electrophoresis similar to myelin basic protein (MBP). MOBP induced experimental autoimmune encephalomyelitis (EAE) in nine of 15 susceptible SJL OlaHsd mice, causing death in two animals, whereas three of 14 BALB/c mice showed mild symptoms of EAE, manifested as transient weakness of hind limbs. In both mouse strains, periventricular infiltrates of mononuclear cells were observed. In addition, both 46 000 MW and 48 000 MW CNP isoforms were shown to be non-encephalitogenic for both mouse strains.

Matrix-assisted laser desorption/ionization mass spectrometry of noncovalent protein-transition metal ion complexes.

Transition metal ion complexes with proteins and peptides are important in many areas of analytical and biological chemistry. We used positive and negative ion MALDI-MS to detect complexes with Cu and Ni ions, and show that the specific and non-specific transition metal ion-peptide complexes can be distinguished by the use of different analytical protocols. The pH dependent stability of these complexes is also reflected in the MALDI data. We further show that triple complexes of peptides or protein with chelated metal ions can be detected efficiently and rapidly by MALDI mass spectrometry. Such triple complexes play an important role in metal chelate affinity chromatography, where histidine containing biopolymers in particular are thought to bind metal-ligand complexes, depending on the oxidation state of the metal and the number of unoccupied coordination sites of the ligand.

Antigen mimicry by an anti-idiotypic antibody single chain variable fragment

For the therapy of cancer patients whose disease is positive for Carcinoembryonic Antigen (CEA), we developed an active specific immunotherapy based on the idiotypic network. The anti-idiotype monoclonal antibody (mAb), 3H1 was generated by immunization of mice with the anti-CEA mAb, 8019. 3H1 mimics CEA both functionally and structurally and acts as a surrogate for CEA. To define the minimum structural requirements for antigen mimicry by 3H1, we constructed plasmid vectors for expression of single chain Fv (scFv) variants of 3H1 in Escherichia coli. Variable heavy (VH) and variable light (VL) chain domains of 3H1 were linked by a 15 amino acid linker (Ln), (Gly 4Ser) 3 in two constructs, VH-Ln-VL and VL-LnVH. Ln was omitted in two constructs, VH-VL and VL-VH. Each of the scFv constructs has a tag of six His [(His) 6 tag] for purification by metal chelate affinity chromatography and detection by enzyme-linked immunoabsorbent assay (ELISA). Comparisons of the binding of 8019 to purified scFv proteins by ELISA and immunoblot experiments showed that only VH-Ln-VL had significant activity. VH-Ln-VL also showed maximum inhibition of binding of 8019 to CEA. Immunization of mice with naked VH-Ln-VL and VH-Ln-VL conjugated to keyhole limpet hemocyanin induced anti-CEA antibodies in mouse sera. Sera from immunized mice inhibited the binding of 8019 to 3H1 as well as CEA. Induction of anti-CEA antibodies in the immunized mice was confirmed by flow cytometric analysis using CEA positive MC-38cea cells. These results demonstrate that for antigen mimicry of 3H1 scFv, the presence of Ln is necessary and the domain order should be VH followed by VL.

Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis

Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens. Since purification of protein from M. tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M. tuberculosis from E. coli cells. Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography. The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E. coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells. The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M. tuberculosis) counterparts. Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.

Characterization of the Nucleic Acid Binding Properties of Tomato Spotted Wilt Virus Nucleocapsid Protein

Tomato spotted wilt tospovirus (TSWV) is the type member of the plant-infecting viruses of the genusTospovirusin the family Bunyaviridae. The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral transcription and replication. Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed inEscherichia coliBL21 (DE3) cells. Full-length N, N amino and carboxy halves, and two N carboxy-terminal regions were expressed and isolated by metal chelate affinity chromatography. The N protein, both of its halves and the extreme carboxy-terminal region, bound cooperatively and irrespective of sequence to radiolabeled single-stranded RNA produced by runoff transcription of clones of either TSWV S RNA or cowpea chlorotic mottle virus RNA3. N protein did not bind to radiolabeled double-stranded TSWV RNA. The density of the synthetic RNase-sensitive N protein–RNA complexes was 1.32 g/ml, similar to the density of authentic Bunyaviridae RNPs. These studies are the first to indicate differences in the nucleic acid binding abilities ofTospovirusandHantavirusnucleocapsid proteins, the only characterized nucleocapsid proteins of the family Bunyaviridae.

Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase.

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.

Expression and purification of recombinant hRPABC25, hRPABC17, and hRPABC14.4, three essential subunits of human RNA polymerases I, II, and III.

Transcription of eukaryotic genes is performed by RNA polymerases I, II, and III, which synthesize ribosomal, messenger, and transfer RNAs, respectively. Eukaryotic RNA polymerases are large macromolecular complexes composed of multiple subunits. Among these subunits, five are shared by all RNA polymerases and are essential for cell growth and viability. Remarkably, the human common subunits are structurally conserved and functionally interchangeable with their yeast homologues and are believed to play an important role in the assembly of the three transcription complexes. To understand the structure and function of human RNA polymerases, we overexpressed the common subunits hRPABC25, hRPABC17, and hRPABC14.4 as hexahistidine fusions in Escherichia coli. The recombinant proteins were purified using metal-chelate affinity chromatography on Ni-NTA resin and gel filtration. Depending on the subunit, the yield was 5-17 mg of purified recombinant protein per liter of culture medium. The purified proteins were of high quality and sufficient quantity for structural studies, as demonstrated by the successful crystallization of hRPABC17 and hRPABC14.4. The expression and purification of the common subunits hRPABC25, hRPABC17, and hRPABC14. 4 will make possible their structural analysis with X-ray crystallography and nuclear magnetic resonance, providing important insights into the structure and function of the three human RNA polymerases.

Improved method for the on-line metal chelate affinity chromatography–high-performance liquid chromatographic determination of tetracycline antibiotics in animal products

An improved on-line metal chelate affinity chromatography–high-performance liquid chromatography (MCAC–HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC–UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 μg kg −1. Limits of detection less than 10 μg kg −1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC–HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

Confirmation of multiple tetracycline residues in milk and oxytetracycline in shrimp by liquid chromatography–particle beam mass spectrometry

A confirmation procedure is described for detection of residues of six tetracyclines in bovine milk, and oxytetracycline in shrimp. Residues are extracted from milk or shrimp tissue using metal chelate affinity chromatography. The extracts are desalted, further concentrated using polymeric solid-phase extraction, and chromatographed on a polymeric reversed-phase column. Analysis is by methane negative ion chemical ionization on a quadrupole mass spectrometer using a particle beam interface. Data are acquired in partial scan mode, monitoring from m/ z 378 to m/ z 480. The procedure was validated with control milk and shrimp, fortified milk (30 ng/ml) and shrimp (100 ng/g), and milk and tissue from animals treated with the drugs.

Molecular characterization of metal-binding polypeptide domains by electrospray ionization mass spectrometry and metal chelate affinity chromatography

Metal ion-binding of synthetic peptides containing HxH and CxxC motifs was investigated by electrospray ionization mass spectrometry (ESI-MS) and metal chelate affinity chromatography. A high affinity of Ni 2+ and Cu 2+ to HxH containing sequences was found. Based on their natural metal ion-binding potential it was possible to include metal affinity chromatography in the purification process of two proteins without using an additional His-tag sequence: ATPase-439, a P type ATPase from Helicobacter pylori and the amyloid precursor protein (APP).

The Primitive Protozoon Trichomonas vaginalisContains Two Methionine γ-Lyase Genes That Encode Members of the γ-Family of Pyridoxal 5′-Phosphate-dependent Enzymes

Methionine gamma-lyase, the enzyme that catalyzes the breakdown of methionine by an alpha,gamma-elimination reaction and is a member of the gamma-family of pyridoxal 5'-phosphate-dependent enzymes, is present in high activity in the primitive protozoan parasite Trichomonas vaginalis but is absent from mammals. Two genes, mgl1 and mgl2, encoding methionine gamma-lyase, have now been isolated from T. vaginalis. They are both single copy, encode predicted proteins (MGL1 and MGL2) of 43 kDa, have 69% sequence identity with each other, and show a high degree of sequence identity to methionine gamma-lyase from Pseudomonas putida (44%) and other related pyridoxal 5'-phosphate-dependent enzymes such as human cystathionine gamma-lyase (42%) and Escherichia coli cystathionine beta-lyase (30%). mgl1 and mgl2 have been expressed in E. coli as a fusion with a six-histidine tag and the recombinant proteins (rMGL1 and rMGL2) purified by metal-chelate affinity chromatography. rMGL1 and rMGL2 were found to have high activity toward methionine (10.4 and 0.67 mumol/min/mg of protein, respectively), homocysteine (370 and 128 mumol/min/mg of protein), cysteine (6.02 and 1.06 mumol/min/mg of protein), and O-acetylserine (3.74 and 1.51 mumol/min/mg of protein), but to be inactive toward cystathionine. Site-directed mutagenesis of an active site cysteine (C113G for MGL1 and C116G for MGL2) reduced the activity of the recombinant enzymes toward both methionine and homocysteine by approximately 80% (rMGL1) and 90% (rMGL2). In contrast, the activity of mutated rMGL2 toward cysteine and O-acetylserine was increased (to 214 and 142%, respectively), whereas that of mutated rMGL1 was reduced to 39 and 49%, respectively. These findings demonstrate the importance of this cysteine residue in the alpha,beta-elimination and alpha, gamma-elimination reactions catalyzed by trichomonad methionine gamma-lyase.

Cobalt resistance in Neurospora crassa: overproduction of a cobaltoprotein in a resistant strain

A cobalt-resistant strain of Neurospora crassa (cor) is 20-fold more resistant to Co when compared with the wild type. DEAE-cellulose and metal-chelate affinity chromatography of cell-free extracts separated cobalt into protein-bound and free ionic fractions. In N. crassa cor about 80% cobalt of cell-free extracts was protein bound while the same in wild type was only 25%. Cobalt content of the protein-bound fraction increased with time and cobalt concentration in the growth medium, and was not influenced by related metal ions. A cobaltoprotein (CBP) which is overproduced in N. crassa cor and constitutes up to 12% of total protein of extracts was purified. CPB is a brown coloured (absorption peaks at 275, 350 and 440nm), small molecular weight glycoprotein (Mr 8200 daltons) with 28 - 30% carbohydrate (mannose). CBP has 70 μg cobalt mg-1 protein. Cysteine, glycine, glutamic acid and aspartic acid are the major amino acid constituents. The role of CBP in cobalt resistance is discussed in relation to other known metalloproteins involved in resistance. © Rapid Science 1998.

Functional expression and purification of histidine-tagged rat renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) cotransporters.

Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/Pi-cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/Pi-cotransport exhibited a Km of Pi of 0.21 mm and an apparent Km of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a Km of SO2-4 of about 0.3-0.4 mm and a Km of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaSi-1 proteins were detected at 55-60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography.

Molecular characterization of the heat-inducible LmSTI1 protein of Leishmania major

We have recently isolated a cDNA encoding the Leishmania major homologue of the yeast stress-inducible protein STI1. Southern blot analyses indicate that this protein is encoded by a single copy gene in L. major and that this gene is highly conserved throughout the Leishmania genus. The STI1 gene is constitutively expressed in both L. major promastigotes and amastigotes however, STI1 transcript levels can be upregulated in promastigotes by a shift in culture temperature from 26 to 37°C. Upregulation of transcript was detectable within 5′ of heat shock and continued to increase for a further 8 h before returning to constitutive levels. In addition, biosynthetic incorporation of [ 35S]methionine followed by immunoprecipitation revealed an increase in the level of nascent STI1 protein synthesized when promastigote cultures were shifted from 26 to 37°C. The L. major STI1 protein and the heat shock proteins Hsp83 and Hsp70 form a salt-sensitive complex in L. major promastigotes as evidenced by co-immunoprecipitation using an antiserum specific for L. major STI1. Furthermore, this complex can be reconstituted in vitro by adding recombinant STI1 containing an amino-terminal histidine tag to promastigote lysate and subsequent purification using metal chelate affinity chromatography.

Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris.

Recombinant Arabidopsis thaliana NADH:nitrate reductase (NR; EC 1.6.6.1) was produced in the methylotrophic yeast Pichia pastoris and purified to near-electrophoretic homogeneity. Purified enzyme had the spectral and kinetic properties typical of highly purified NR from natural plant sources. Site-directed mutagenesis altering several key residues and regions was carried out, and the mutant enzyme forms were expressed in P. pastoris. When the invariant cysteine residue, cysteine-191, in the molybdo-pterin region of the A. thaliana NIA2 protein was replaced with serine or alanine, the NR protein was still produced but was inactive, showing that this residue is essential for enzyme activity. Deletions or substitutions of the conserved N terminus of NR retained activity and the ability to be inactivated in vitro when incubated with ATP. Enzyme with a histidine sequence appended to the N terminus was still active and was easily purified using metal-chelate affinity chromatography. These results demonstrate that P. pastoris is a useful and reliable system for producing recombinant holo-NR from plants.