Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

A simple method for the purification of murine corticosteroid binding globulin: characterization of a monomer

A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH2-terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (Mr) of murine CBG was determined to be 52,000 and the sedimentation constant S20 degrees, w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15-3.25 and two between pH 3.40-3.50.     

Affinity chromatography of the muscarinic acetylcholine receptor

A novel compound, 3-(2'-aminobenzhydryloxy)-tropane (ABT), and an ABT-agarose gel were synthesized and used for the purification of solubilized muscarinic receptors. ABT had a high affinity with an apparent dissociation constant (Kd) of 7 nM for the muscarinic receptors solubilized from the porcine brain by digitonin. An ABT-agarose gel was prepared by coupling ABT with epoxy-activated Sepharose 6B, and the degree of substitution to the gel was determined to be 4-5 mumol/ml of the gel by UV absorption spectrum. During affinity chromatography using 10 ml of the ABT-agarose gel and 100 ml of the digitonin-solubilized preparation, 70% of muscarinic receptors were adsorbed to the gel, in marked contrast with the adsorption of only 2% of proteins. Approximately 25% of muscarinic receptors applied to the gel were eluted biospecifically with 1 mM muscarinic ligands. The purified fraction showed a high affinity for [3H]quinuclidinyl benzylate with a Kd of 0.4 nM and similar specificity for muscarinic ligands to that of unpurified soluble receptors. The protein concentration of the purified fraction was too low to be determined accurately, but very approximately a purification of 10(3)-fold was indicated.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.     

Possibility of shape conformers of the protein inhibitor of the cyclic adenosine monophosphate dependent protein kinase.

The heat-stable, protein inhibitor of the cyclic adenosine monophosphate (cAMP) dependent protein kinase [Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E., & Krebs, E (1971a) J. Biol. Chem. 246, 1977-1985] has been purified to homogeneity from rabbit skeletal muscle by preparative electrophoresis. Employing a more sensitive assay system, we detected multiple charged forms of the inhibitor on diethylaminoethyl chromatography; the form that has been further characterized is the predominant species in skeletal muscle comprising greater than 70% of the total. The apparent molecular weight of the protein inhibitor, as determined by Sephadex G-75 gel exclusion chromatography, is 22 000 in initial cellular extracts and at all stages during the purification prior to the final purification step of preparative gel electrophoresis, after which the homogeneous protein exhibits a molecular weight of 11 000. These two forms are designated I and I', respectively. The I form migrates with an apparent molecular weight of 10 000 on nondenaturing gel electrophoresis and of 10 500-11 500 on sodium dodecyl sulfate (NaDodSO4) gel electrophoresis; the I' form migrates with an apparent molecular weight of 6500-8300 on NaDodSO4 electrophoresis and has a minimum molecular weight of 10 400 by amino acid analysis. Taking into account the anomalous behavior displayed by low molecular weight proteins with the various techniques employed, we suggest that the I and I' forms of the protein inhibitor may represent shape conformers.     

Purification of human placental trophoblast interferon by two-dimensional high performance liquid chromatography

Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the beta-type (75%) and relatively low levels of the alpha-type (25%). A two-step high performance liquid chromatographic procedure ("two-dimensional HPLC") has been developed for the complete purification of the placental trophoblast interferon beta (tro-IFN-beta) from serum-containing culture supernatant. The method involved a combination of high performance liquid affinity chromatography (HPLAC) on Cibacron Blue 3GA immobilized on an activated pressure stable macroporous synthetic polymer, 2-hydroxyethyl methacrylate vinyl sulphone (HEMA-BIO 1000 VS), as the first dimension and reversed-phase high performance liquid chromatography (RP-HPLC) on Separon SGX C-18 as the second. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments showed that the tro-IFN-beta was present as a 24 kDa protein. Densitometric scanning analysis of Coomassie-stained gel revealed the purity of the final preparation to be greater than 99%. The purified tro-IFN-beta had a specific activity of 1.03 x 10(8) IU/mg of protein and the overall recovery was 81% of the total IFN-beta activity in the crude preparation and 61% of the total IFN activity.     

Affinity purification of retinoic acid-binding proteins using immobilized 4-(2-hydroxyethoxy)retinoic acid

An affinity chromatographic matrix that purifies cellular retinoic acid-binding protein to near homogeneity from rat testes cytosol has been developed. The three-step procedure includes an acid precipitation, a batch treatment with CM Bio-Gel, and affinity chromatography on 4-(2-hydroxyethoxy)retinoic acid coupled to epoxy-activated Sepharose 6B. The binding protein was purified approximately 8500-fold based on total soluble testicular protein and with a recovery in excess of 80%. In addition, further enhancement of the purity of the protein can be attained by size-exclusion HPLC to increase purification to 21,000-fold. The recovered protein has an apparent M(r) 14,300 as determined by size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is isolated in the apo-form and retains its ability to bind retinoic acid as evidenced by the binding of [3H]retinoic acid. An apparent retinoic acid-binding protein of M(r) 18,000 has also been isolated from rat testes nuclei by the affinity chromatography step. The affinity phase has been used for 6 months without any detectable loss in its ability to purify cellular retinoic acid-binding protein.     

Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium

A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.     

Purification and physicochemical characterization of murine T cell replacing factor (TRF)

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.     

Isolation, purification and study of the properties of pyruvate kinase from the bovine adrenal cortex

Pyruvate kinase (ATP: pyruvate 2-0-phosphotransferase, EC 2. 7. 1. 40) from bovine adrenal cortex was purified 243 fold. The whole purification procedure included ammonium sulphate fractionation, heat treatment, Sephadex HW-55 chromatography and phosphocellulose chromatography. The specific activity of the preparation is 15.6 U/mg at 30 degrees C, the yield--36%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. The enzyme displayed a hyperbolic saturation curve with respect to P-enolpyruvate. The apparent Km for this substrate was 0.55 X 10(-4) M, pH optimum--6.8-7.0. K+ concentrations above 0.1 M inhibit the enzyme.     

A rapid, large scale purification procedure for gibbon interleukin 2

Interleukin 2 (IL 2) was purified from the conditioned medium of a gibbon T cell line, MLA144, which releases IL 2 constitutively. The IL 2 was obtained free of contaminating proteins by a simple process consisting of an initial batch purification on trimethylsilyl-controlled pore glass followed by reversed phase high pressure liquid chromatography. Overall recovery of IL 2 activity ranged from 70 to 100% of initial activity and yielded 2 X 10(6) or greater units of IL 2 per 15 liters of serum-free MLA144 conditioned medium. The specific activity of purified IL 2 ranged from 0.5 to 1 X 10(8) IL 2 U/mg protein. The purified IL 2 showed four molecular species when analyzed by two-dimensional isoelectric focusing-SDS-polyacrylamide gel electrophoresis. Each of the four molecular forms was active in the bioassay for IL 2 activity. Three molecular forms had apparent m.w. of 16,000 but different isoelectric points of 5.9, 6.3, and 6.7. One molecular form had an apparent m.w. of 15,000 and an isoelectric point of 7.2. The most abundant form of IL 2 had an apparent m.w. of 16,000 and an isoelectric point of 6.3. The purified IL 2 supported the growth of IL 2-dependent lymphocytes to a greater extent than did the same level of crude IL 2-containing MLA144 conditioned medium. The ability to purify large amounts of IL 2 by a rapid and efficient procedure will be of great help in both biochemical and immunologic studies of this lymphokine.     

Purification of the muscarinic acetylcholine receptor from porcine brain

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.     

Purification of nitric oxide synthase from rat macrophages.

Nitric oxide (NO) synthase (EC 1.14.23) has been purified to apparent homogeneity from rat macrophages. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and gel filtration chromatography on a Superose 12 HR 10/30 column. The apparent molecular weight is 300,000 by gel filtration. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrates as a single protein band with Mr = 150,000. The purified enzyme is colorless, and an absorption maximum is observed at 280 nm. The half-life of the enzyme activity is 6 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-L-biopterin, and dithiothreitol. Although the cerebellar and endothelial enzyme require Ca2+ and calmodulin, these are not required by the macrophage enzyme. The macrophage nitric oxide synthase (an inducible enzyme) seems to be different from the cerebellar and endothelial enzyme (a constitutive enzyme).     

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

The lipid transport system in the mouse, Mus musculus: isolation and characterization of apolipoproteins B, A-I, A-II, and C-III

Four of the principle apolipoproteins of murine serum have been isolated and characterized. On the basis of their physicochemical properties, they are homologous with the human and rat apoA-I, A-II, B, and C-III. The group of apolipoproteins of middle to low molecular weight, i.e., A-I, A-II and C-III, were separated from the protein moiety of high density lipoproteins (HDL) by gel filtration chromatography, followed by electrophoresis in alkaline-urea polyacrylamide gel with electrophoretic elution. Murine apoA-I, the major protein of HDL (60-80%) displayed an Mr of approximately 27,000, and was polymorphic (four prominent isoproteins with isoelectric points in the range of pH 5.5-5.7). The amino acid profiles of mouse, rat, and human apoA-I generally resembled each other, the former being distinguished by a content of one isoleucine residue per mole. Amino terminal sequence analysis revealed marked homology between the mouse, rat, dog, and human proteins; mouse and rat apoA-I differed at residues 9 and 18 with potential dissimilarities at residues 5 and 15, while the murine and canine sequences were distinct at residues 6, 9, 13, 15, and 30. Apolipoprotein A-II was a monomer, exhibiting an Mr approximately 11,000 in SDS gels; in addition, it was polymorphic (three apparent isoproteins with pI in the pH range 5.05-5.2), and resembled its human and rat counterparts in amino acid composition. ApoC-III, an acidic peptide of pI 4.74 and of Mr approximately 9,600, possessed an amino acid composition very like that of the homologous human and rat proteins. The homology of mouse apoC-III with the human protein was confirmed by NH2-terminal sequence analysis, which revealed identical amino acids in six positions (1, 2, 4, 8, 9, and 13). As shown earlier (Camus et al. 1983. J. Lipid Res. 24: 1210-1228), two forms of immunologically reacting apoB predominated in mouse VLDL and LDL. After isolation of these lipoproteins in the presence of 1 mM PMSF, the apparent sizes of the high and low Mr forms, apoBH and apoBL, were in the ranges approximately 400,000-530,000 and approximately 250,000-280,000, respectively, according to the SDS gel system. We observed that inclusion of 1 mM PMSF was essential to retard degradation of the high Mr form apoBH. The murine B proteins were isolated from apoVLDL and apoLDL by gel filtration chromatography on Sephadex G150 in anionic detergent, and displayed apparent Mr values of 460,000 (apoBH) and 250,000 (apoBL) in 3% SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

A large-scale purification of beta-glucuronidase from human liver by immunoaffinity chromatography.

We intend to purify beta-glucuronidase from human liver in a large quantity in order to facilitate the study of its biochemical structure and pathophysiologic roles in cholelithiasis and carcinogenesis. The initial purification procedure involved: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation of protein at 56 degrees C, (4) gel filtration with Bio-Gel P-300 gel, (5) anion exchange chromatography with DEAE agarose, (6) cation exchange chromatography with CM agarose, and (7) hydroxyapatite chromatography (overall yield, 1%; overall purification, 169X). The final product was used to immunize rabbits and BALB/c mice for production of polyclonal and monoclonal antibodies, respectively. The antibodies, mainly IgG, were purified by using gamma-Protein A agarose column chromatography. The purified IgG, after periodate oxidation, was coupled to hydrazide gel by formation of a stable covalent hydrazone bond linkage. The new purification procedure involved the initial first three steps, followed by (4) polyclonal IgG immunoaffinity chromatography and (5) monoclonal IgG immunoaffinity chromatography (overall yield, 6.1%; overall purification, 3720X). Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by electroelution. It is concluded that beta-glucuronidase constitutes 0.016 mg per gram of wet liver tissue and can be obtained on a large scale in a highly purified form within a 2-day cycle.     

Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.     

Purification of the Ah receptor from C57BL/6J mouse liver

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.     

Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.     

Purification of BBF, a DNA-binding protein recognizing a positive cis-acting element in the mouse alpha 1(III) collagen promoter.

A positive cis-acting element, the B element, located between -83 and -61 in the mouse alpha 1(III) collagen promoter, binds a factor present in nuclear extracts of NIH 3T3 fibroblasts and HeLa cells. We have purified this factor using ion exchange chromatography, sequence-specific DNA affinity chromatography, and sodium dodecyl sulfate-polyacrylamide gel fractionation. The DNA sequence used for the affinity chromatography was a single-base substitution in the B element that increased the stability of the B element-protein complex by 50%. Purification of the B element-binding factor (BBF) by DNA affinity chromatography resulted in the apparent loss of most or all of the DNA-binding activity of this factor. The DNA-binding activity could, however, be reconstituted by combining two chromatographic fractions: the high-salt eluate and the column flow-through. When the partially purified high-salt eluate was size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with subsequent renaturation of gel fractions from guanidine HCl, the purified BBF (apparent molecular weight of about 95,000) bound to the B element with high affinity. These results suggest that during DNA affinity purification of BBF a factor that inhibits BBF DNA binding was co-eluted with BBF. This inhibition of BBF DNA binding was reversed by the addition of the DNA affinity column flow-through. The binding of BBF to the B element of the mouse alpha 1(III) collagen promoter is therefore an apparently complex process involving interactions between BBF and other protein factors.