Production and Purification of a Staphylococcus epidermidis Bacteriocin

Liquid cultures of Staphylococcus epidermidis 1580 contained rather small amounts of a bacteriocin, staphylococcin 1580, which was found both in the supernatant fluid and in the cell pellet. It could be extracted from the cells with 5% NaCl solution. The staphylococcin production could not be induced by ultraviolet irradiation or treatment with mitomycin C. Bacteria grown on semisolid medium produced a much larger amount of the compound with a high specific activity. The staphylococcin was purified by ammonium sulfate precipitation, ultracentrifugation, and chromatography on Sephadex columns. The purified material was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was between 150,000 and 400,000. The bacteriocin was composed of protein, carbohydrate, and lipid and consisted of subunits exhibiting a molecular weight of about 20,000.     

Nature and Properties of a Staphylococcus epidermidis Bacteriocin

Staphylococcin 1580, produced by Staphylococcus epidermidis 1580, consisted of 41.8% protein, 34% carbohydrate, and 21.9% lipid. In the protein fraction, the acidic amino acids, glutamic and aspartic acid, and the neutral amino acids, glycine and alanine, predominated. Neutral sugars consisted of glucose, galactose, and fucose in a molar ratio of 6:3:1. The purified bacteriocin was not inactivated by heating for 15 min at 120 C in the presence of 0.5% serum albumin and was stable in the pH range from 3.5 to 8.5. The compound was sensitive to the action of the proteolytic enzymes trypsin, Pronase, and chymotrypsin. All gram-negative bacteria tested were resistant; a large number of gram-positive bacteria were sensitive to staphylococcin 1580 action. Growth of stable staphylococcal L-forms was inhibited by the bacteriocin to the same extent as their parent strains. The staphylococcin was adsorbed to cell walls, cell membranes, and resistant cells. The effect of staphylococcin 1580 appeared to be bactericidal but not bacteriolytic.     

RNA-Stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3

Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.     

Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates

Ubiquitin-lysozyme conjugates have been used as substrates to identify an ATP-dependent protease from rabbit reticulocyte lysates. The enzyme, which has been partially purified by DEAE chromatography and glycerol gradient centrifugation, has an apparent molecular weight greater than 600,000 based on sedimentation and gel filtration. Whereas it degrades conjugated lysozyme molecules in the presence of ATP, the protease does not degrade free lysozyme molecules even upon addition of ubiquitin, lysozyme-ubiquitin conjugates, and ATP. Degradation of lysozyme conjugates is independent of added ubiquitin and occurs in fractions incapable of ubiquitin conjugation. Proteolysis is maximal at pH 7.8, inhibited by hemin, N-ethylmaleimide, or aurintricarboxylic acid, and proceeds with an apparent Arrhenius activation energy in the range of 27 +/- 5 kcal/mol. These properties are similar to those observed for the degradation of lysozyme conjugates in lysates indicating that the partially purified protease catalyzes the "second" ATP-utilizing reaction identified previously (Hough, R., and Rechsteiner, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 90-94; Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1619-1623; Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585).     

链霉菌Z314的分类鉴定及其转化产物普伐他汀的纯化研究

从土壤中筛选得到1株可转化美伐他汀(compactin)生产普伐他汀(pravastatin)的放线菌Z314,根据形态观察、培养特征、生理生化鉴定以及16S rRNA序列分析,初步判定该菌株为链霉菌属中的黄质产色链霉菌(Streptomyces xanthochromogenes).经发酵条件优化,该菌株转化美伐他汀生产普伐他汀的产量可达1580mg/L,转化率为49.45%.发酵液经中空纤维素柱过滤、大孔树脂吸附和HPLC纯化,普伐他汀的回收率为45.06%,纯度为99.02%,为进一步产业化开发奠定了基础.     

Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

Background

The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable san-1(ok1580) deletion mutant.

Results

The san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein.

Conclusion

Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.     

重组人胰岛新生相关蛋白的表达、纯化及免疫活性研究

摘要 目的：对胰岛新生相关蛋白（Islet neogenesis associated protein ,INGAP）进行表达、纯化,并检测其免疫活性。方法： INGAP基因片段插入表达载体pET22b(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,Heparin Agrose亲合柱层析为第一步纯化,Superdex75凝胶过滤层析作为第二步精细纯化,HPLC测定INGAP蛋白的浓度,将纯化的INGAP蛋白经注射途径免疫家兔,制备兔抗INGAP血清,采用免疫双扩、ELISA及Western Blot分析INGAP的免疫活性。结果INGAP以包涵体形式表达,表达产量高达总菌体蛋白的40%左右,经Heparin Agrose亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.81%,表达及纯化的INGAP具有良好的免疫活性。     

Purification of 125I-labeled succinyl cyclic nucleotide tyrosine methyl esters by high-performance liquid chromatography

Carrier free 125I-labeled succinyl cyclic adenosine monophosphate (ScAMP) and succinyl cyclic guanosine monophosphate (ScGMP) tyrosine methyl esters (TME) were purified by reversed phase high-performance liquid chromatography (HPLC) or descending paper chromatography. Using an isocratic buffer for HPLC, mono-ScAMP-125I-TME and mono-ScGMP-125I-TME were eluted from a C18 column at 8.9 and 6.9 min, respectively. Both of the mono-iodinated radioligands were completely separated from their noniodinated precursors and other iodinated products. The radioligands purified by HPLC or paper chromatography were used for the radioimmunoassay (RIA) of cAMP and cGMP. Cyclic AMP or cGMP inhibited binding of the HPLC purified radioligands at three- to fivefold lower concentrations than the paper chromatography purified radioligands. The sensitivity of the RIA decreased with time if paper chromatography purified radioligands were used, but remained stable for 4 months if the HPLC purified compounds were used, even with storage at 4 degrees C. We attribute these results to better purification of radioligands by the HPLC than by the paper chromatography. Using optimal conditions the HPLC method takes only 10 min and results in a high yield (greater than 95%) of added 125I into the monoiodinated products.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

The role of the carbohydrate moiety on the size heterogeneity and immunologic determinants of human testosterone-estradiol-binding globulin

Human testosterone-estradiol-binding globulin (hTeBG) has been purified to apparent homogeneity by several laboratories using procedures which, in most instances, were labor intensive. In this report, hTeBG was purified from pregnancy serum by a newly developed two step procedure involving sequential affinity chromatography and ion-exchange high performance liquid chromatography (ion-exchange HPLC). The purity of the final product was confirmed by silver stained SDS-polyacrylamide gel and reverse phase HPLC monitored at 206 nm. hTeBG purified by ion-exchange-HPLC maintained binding activity by Dextran coated charcoal (DCC) assay and size heterogeneity on SDS-polyacrylamide gels which were indistinguishable from those of the proteins purified by conventional chromatography. Removal of the carbohydrate moiety from the molecule by both enzymatic and chemical treatment reduced the apparent molecular size and eliminated lectin binding of hTeBG subunits. Deglycosylation did not, however, abolish or alter the distribution of the protomeric forms of this subunit. We conclude that hTeBG is a dimer whose monomer exhibits two protomeric forms which is not a result of carbohydrate heterogeneity. In addition, disialylated and deglycosylated hTeBG exhibited antigenic determinants identical to the native protein.     

Androgen metabolism by human peritoneal macrophages

Peritoneal macrophages (PM) were obtained by peritoneal dialysis from a regularly menstruating woman with renal failure. Macrophages (10(6) cells) were incubated at 37 degrees C for various periods of time (0-4 hr) in the presence of 14C-androstenedione or 3H-androstenedione and various concentrations (0.06-5.06 microM) of nonradiolabeled androstenedione (A). Testosterone (T) formed was purified by column chromatography, thin layer chromatography, acetylation, and recrystalization to constant 3H:14C ratios. The rate of formation of T from A was linear for nearly 2 hr. Conversion of A to T was linear at cell numbers in the incubation up to 1 x 10(6). The formation of T from A followed Michaelis-Menten kinetics at concentrations of A between 0.06 and 5.06 microM. The apparent Km of the enzyme for A was 0.75 microM and the Vmax for T formation from A in these cells was 33.9 pmol x hr-1 x 10(6) cells-1. PM were obtained also from normal patients (n = 6) and patients with endometriosis (n = 5). The rate of T synthesis from A in PM obtained from patients with endometriosis [527 +/- 263 pmol x hr-1 x 10(6) cells-1 (mean +/- SEM, n = 5)] was similar to that observed in PM obtained from normal patients [518 +/- 226 pmol x hr-1 x 10(6) cells-1 (mean +/- SEM, n = 6)]. We observed a near 30-fold variation in the rate of formation of T from A by PM obtained from different individuals (range 54 to 1580 pmol x hr-1 x 10(6) cells-1). Further study is needed to elucidate the physiologic significance of PM androgen metabolism and its relationship to reproductive function.     

Separation of lymphocyte mitogen from lymphotoxin and experiments on the production of lymphotoxin by lymphoid cells stimulated with the partially purified mitogen: a possible amplification mechanism of cellular immunity and allergy.

A mitogenic factor (MF) from guinea pig lymph node cells (LNC), which was produced by stimulating immune LNC with the specific antigen, ovalbumin, was partially purified by the sequential use of Sephadex G-200 gel filtration. CM-cellulose ion exchange chromatography, DEAE-cellulose ion exchange chromatography, and polyacrylamide disc gel electrophoresis. The product was purified at least 100-fold with regard to protein content. In addition, the purified MF was functionally pure with respect to the absence fo lymphotoxin (LT), another guinea pig lymphokine.     

Purification and characterization of xylanase from a thermophilic Streptomyces sp. K37

Extracellular xylanase (EC 3.2.1.8) from Streptomyces sp. K37 was purified 33.53 by ultrafiltration and cation exchange chromatography followed by gel filtration chromatography. The optimum pH and temperature for purified xylanase were found to be pH 6.0 and 60 degrees C. The Km and V(max) values of the purified xylanase were 15.4 mg ml(-1) and 0.67 micromole reducing sugar min(-1) ml(-1). High performance liquid chromatography (HPLC) gel filtration of the purified xylanase eluted xylanase activity as a peak corresponding to the molecular weight of about 24.3 kDa while the molecular weight determined by SDS-PAGE was found to be 26.4 kDa. The purified xylanase of Streptomyces sp. K37 was found to be endoxylanase and non arabinose liberating enzyme and was highly glycosylated (73.97%).     

Purification and characterization of the D2 cell adhesion protein: Analysis of the postnatally regulated polymorphic forms and their cellular distribution

The developmentally regulated, D2 cell adhesion protein has been purified from 10–12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5–1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (>200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.     

嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立

【目的】通过定点突变确定嗜酸热脂环酸杆菌中甘露聚糖酶的活性催化位点。【方法】根据序列比对和GH53家族的结构信息选择可能的催化活性位点,利用重叠PCR法构建定点突变体,采用薄层层析(TLC)法和3,5-二硝基水杨酸(DNS)法检测各酶蛋白活性。【结果】通过重叠PCR法成功构建了7个位点的突变体,其中第150和159位的氨基酸突变对活性改变甚少或几乎没有,而第151和231位谷氨酸的羧基基团的改变以及双位点突变体E2Q则导致其对各种底物催化活性的丧失,说明位于β4和β7折叠的C末端的E151和E231的羧基基团作为功能基团参与了催化反应。【结论】E151和E231分别是新型甘露聚糖酶AaManA的酸碱催化位点和亲核催化位点。     

Purification of boiling-soluble antifreeze protein from the legume Ammopiptanthus mongolicus

A boiling-soluble antifreeze protein (AFP) was purified from the winter leaves of Ammopiptanthus mongolicus an evergreen legume species surviving in the cold desert of northwest of China. Purification was achieved by using a procedure consisting of a heat treatment step followed by consecutive chromatography, including ion-exchange chromatography (DEAE-Cellulose 52, Source 15Q), molecular exclusion chromatography with Sephacryl S300, and hydrophobic interaction chromatography (Poros 20HP2). This AFP showed thermal hysteresis activity and could modify the normal growth of ice crystals. The thermal hysteresis activity (THA) of this purified antifreeze protein is 0.15 degrees C at a concentration of 10 mg/mL, and its molecular mass is approximately 28 kD by SDS-PAGE analysis.     

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.     

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin.     

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.     

Purification of a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) by chromatography and its use as an affinity ligand

An ammonium sulphate fraction (20–60%) of bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) was purified by affinity chromatography to give 6.59-fold purity with 81.48% yield. The same ammonium sulphate fraction was also subjected to ion exchange chromatography and was purified 4.28-fold with 75.95% yield. The ion exchange fraction was subjected to gel filtration and the inhibitor was purified to 6.67-fold with 67.36% yield. Further sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the homogeneity of purified amylase/trypsin inhibitor obtained through affinity, ion exchange and gel chromatography. The molecular weight of the inhibitor was found to be 14 kDa. This purified inhibitor was used as affinity ligand for the purification of a commercial preparation of pancreatic amylase.