Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase

A method for the purification of highly pure and active Escherichia coli RNA polymerase holoenzyme is described. This method is simple, reproducible, and can be performed at room temperature. The procedure involves the high-performance liquid chromatography of a partially purified RNA polymerase sample on a Mono Q ion-exchange column. Under the conditions used, RNA polymerase holoenzyme is well separated from the core RNA polymerase and other impurities. The purified RNA polymerase contains virtually no impurities as judged by SDS-polyacrylamide gel electrophoresis. The purified RNA polymerase holoenzyme contains the sigma 70 subunit in stoichiometric amounts and is at least 90% active.     

Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.

A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.     

Purification of a full-length recombinant glucocorticoid receptor

We described a novel purification method for a recombinant glucocorticoid receptor (GR) in detail. The purification procedure consists of sequential chromatographies using common ion-exchange columns (Mono Q and Mono S). This procedure is based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH. The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors.     

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

Purification of Dictyostelium myosin II heavy chain kinase A based on the increase in negative charge accompanying hyperphosphorylation

The initial step in the purification of Dictyostelium myosin II heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg2+ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 M KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M(r) greater than 700,000).     

Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor

Human macrophage differentiation inducing factor (DIF) can induce differentiation of human myeloid leukemic cells into macrophage-like cells in vitro. A procedure is described for purification of DIF from serum-free human monocytic leukemia THP-1 cell-conditioned medium. The procedure included concentration of a conditioned medium by ultrafiltration, lentil lectin-Sepharose affinity chromatography, and fast protein liquid chromatography using Mono S and Mono Q. DIF-A of pI 9.0 and DIF-B of pI 8.8 were obtained after a final purification with a Mono Q column, and both DIF gave a single peak with a molecular weight of approximately 51,000 determined by gel chromatography. NH2-terminal amino acid analysis of DIF-A showed a noticeable homology with murine leukemia inhibitory factor. Human DIF-A was found to induce maturation of human and murine leukemic cells into both macrophage-like cells with nitro blue tetrazolium reducing activity and phagocytic cells, but was found to suppress proliferation of these leukemic cells.     

Purification of Dictyostelium myosin II heavy chain kinase a based on the increase in negative charge accompanying hyperphosphorylation

The initial step in the purification of Dictyostelium myosin 11 heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg²⁺ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 m KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M_r greater than 700,000).     

Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats.

A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously with heat-killed Propionibacterium acnes and 5 days later with Escherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by 2',5'-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors NG-monomethyl-L-arginine and N omega-nitro-L-arginine at 80 and 800 microM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independent isozyme on Mono Q column chromatography. The purified calmodulin-independent isozyme was converted to a calmodulin-dependent isozyme by EDTA and EGTA. Calmodulin blot analysis using 125I-calmodulin showed that the two isozymes bound calmodulin equally efficiently.     

Purification of NAD glycohydrolase from Agkistrodon acutus venom

NAD glycohydrolase (NADase) from Agkistrodon acutus venom was purified to electrophoretic homogeneity by a fast, reproducible 3-step procedure including Q Sepharose Fast Flow, Superdex 75, and Mono S column chromatography. This new procedure gave a 15.6-fold purification with a recovery yield of 7.9% and a specific activity of 12.8 units/mg.     

利用FPLC提纯抗苯丙氨酸羟化酶抗体的Fab及其轻链和重链的分离

 IgGPH_7经木瓜蛋白酶消解产生Fab和Fc,在FPLC仪上,分别用MonoQ,Superose_(12),MonoS柱提纯Fab。提纯的Fab经还原和烷基化后,再用琥珀酸酐修饰,然后用Mono Q柱分离得到轻链和重链。最后通过SDS凝胶电泳和N端顺序测定,以鉴定它的纯度和轻、重链。     

Simultaneous purification of DNA topoisomerase I and II from eukaryotic cells

We have developed a procedure for the simultaneous purification of DNA topoisomerase I and II from calf thymus. Both enzymes were first extracted from isolated nucleoprotein complexes. After batchwise chromatography on hydroxylapatite the two enzyme activities were separated on a FPLC phenylsuperose column. The enzymes were further purified by a second chromatography on phenylsepharose (topo I) or FPLC Mono Q (topo II). The purification can be finished within three days, yielding 0.5-1.0 mg quantities of homogeneous, enzymatic active preparations of the two proteins from 200 g of starting material.     

The use of immobilized methylchymotrypsin for the purification of human and sheep alpha-1-proteinase inhibitor (alpha(1)-PI)

alpha(1)-proteinase inhibitor was isolated from albumin fractions of human and sheep plasma using a new method of purification using affinity chromatography on immobilized methylchymotrypsin in the presence of 5 M NaCl. The inhibitor was finally polished to homogenity either by chromatography on a Mono Q or a Sephacryl S-200 HR column. The presented method makes it possible to recover alpha(1)-proteinase inhibitor which has been added to cow milk.     

Behaviour of vanadate and vanadium-transferrin complex on different anion-exchange columns. Application to in vivo 48V-labelled rat serum

The behaviour of free [48V]vanadate and [48V]vanadium-transferrin complex was investigated on five different anion-exchange columns (Mono Q 5/5 HR, Hitrap Q HP, Sepharose Q FF, Sepharose DEAE FF and Hitrap Q XL). The recovery of both V-compounds was quantitative. The peak shape and retention time of vanadate varied according to the type of column. The vanadium-transferrin complex also showed different elution patterns depending on the type of column. Especially in case of the Sepharose Q FF, Mono Q 5/5 HR and Hitrap Q XL columns the vanadium-transferrin binding was degraded during elution on the column. The results clearly prove that care should be taken as to the choice of column for speciation purposes of vanadium compounds in order to prevent various artefacts showing up in the chromatograms. A Hitrap Q HP column was used to fractionate different vanadium compounds in rat serum.     

Synthesis and rapid purification of UDP-[6-3H]galactose

UDP[6-3H]galactose of high specificity can be obtained by oxidation of the C-6 hydroxymethyl group of UDP-galactose by galactose oxidase and subsequent reduction by sodium borotritide. One-step purification of the nucleotide sugar involves anion-exchange chromatography on a Pharmacia Mono Q column. Radiolabeled UDP-N-acetylgalactosamine can also be synthesized and purified by this procedure. Both nucleotide sugars can be used for sugar incorporation studies using the appropriate glycosyltransferase.     

Purification and characterization of DNA polymerases from Bacillus species.

DNA polymerases from Bacillus stearothermophilus, Bacillus caldotenax, and Bacillus caldovelox were purified by chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose and obtained in high yield. The enzyme preparations are free of exo- and endonuclease activities. Additional purification steps, e.g., hydrophobic interaction chromatography and chromatography on a Mono Q column or sucrose density gradient centrifugation, are needed to obtain the enzymes in the form of homogeneous 95-kDa proteins. Each of the three organisms possesses a major DNA polymerase activity comparable to DNA polymerase I. The enzymes require Mg2+ (10 to 30 mM) for optimal activity, although 0.4 mM Mn2+ could substitute for magnesium. The optimal reaction temperatures were lowest in B. stearothermophilus (60 to 65 degrees C) and about equal in B. caldovelox and B. caldotenax (65 to 70 degrees C). The thermal stabilities of the enzymes increased in the same order. The DNA polymerase from Thermus thermophilus was isolated for comparison by using a similar procedure. The enzyme was obtained as a homogeneous 85-kDa protein that was also free of exo- and endonucleolytic activities.