Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

大肠杆菌热稳定性肠毒素的分离纯化和氨基酸顺序测定

上海市儿童医院婴儿腹泻病患中分离到产肠毒素的菌株,该菌株只产热稳定性肠毒素,经CAD-44树脂吸附,SephadexG-25凝胶过泸,DEAE-Seharose CL-6B离子交换层析,反相高压液相层析分离纯化后,用手工固相DABITC/PITC双偶合Edman降解法测定其氨基酸顺序为:Asn·Ser·Ser·Asn·Tyr·Cys·Cys·Gly·Leu·Cys·Cys·Asn·Pro·Ala·Cys·Thr·Gly·Cys·Tyr。     

Purification of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli strain 080

Purification studies of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 degrees C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54,000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera.     

Purification and properties of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600

A preparative scale method for isolation of highly purified phenylalanyl-tRNA synthetase from E. coli MRE-600 was developed. It consists of cell destroying, nucleic acid precipitation with streptomycine sulfate, fractionation with ammonium sulfate followed by chromatography on different carriers (Sephadex G-200, DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite). The mode of cell destroying was found to affect the process of the further enzyme purification. The phenylalanyl-tRNA synthetase was purified 540-fold, with recovery being 20.6% and the specific activity - 540 units per mg protein. The enzyme content in the purified preparation was 80-90% judging by electrophoresis in PAAG. The molecular weights of the subunits determined by electrophoresis under denaturative conditions were found to be 102,000 +/- 4000 (beta) and 42,000 +/- 2000 (alpha). The molecular weight of the native enzyme determined by gel filtration through Sephadex G-200 and electrophoresis at varied concentrations of polyacrylamide was found to be 340,000 +/- 20,000. The Km values for tRNA, ATP and phenylalanine in the aminoacylation reaction are equal to 5.4 X 10(-7) M, 1,9 X 10(-4) M, and 3.7 X 10(-6) M, respectively.     

Guanylate cyclase in Escherichia coli. I. Purification of the enzyme and evidence for an inhibitor]

Guanylate cyclase activity is present in crude E. coli extract. Guanylate cyclase has been purified 3500 fold from this extract, through ammonium sulfate fractionation, DEAE-cellulose chromatography. Sephadex G-75 gel-filtration and polyacrylamide gel preparative microelectrophoresis. During the purification a guanylate cyclase inhibitor has been separated.     

Purification and characterization of streptomycin 6-kinase, an enzyme implicated in self-protection of a streptomycin-producing micro-organism

Streptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5'-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mM and 0.4 mM, respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin.     

Messenger RNA of the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi. Isolation and properties.

Polysomes specifically synthesizing the large subunit of ribulose-1,5-bisphosphate carboxylase were isolated from Chlamydomonas reinhardi cells by the indirect immunoprecipitation method. Electrophoretic analysis showed that the immunoprecipitated polysomes were of chloroplast origin. The mRNA coding for the large subunit which was purified from immunoprecipitated polysomes migrated at the 19-S position on sucrose density gradients, and its molecular weight was estimated to be 7.3 x 10(5) by acid-urea/agarose gel electrophoresis. The mRNA was translated in vivo with a cell-free protein-synthesizing system derived from Escherichia coli to give full-length large-subunit polypeptides.     

A quantitative analysis of DNA extraction and purification from compost

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.     

新的端粒酶活性抑制蛋白LPTS-L的制备

LPTS基因是利用定位候选克隆策略克隆的一个新的肝相关候选肿瘤抑制基因。LPTS基因编码一个全长为328氨基酸的蛋白质(LPTS-L),该蛋白具有抑制细胞端粒酶活性的功能。为了进一步研究LPTS-L蛋白的结构与功能,利用DNA重组技术,将LPTS-L的cDNA克隆到表达载体pET-24a中构建重组克隆pET-24-LPTS,并在大肠杆菌BL-21中进行融合表达,获得可溶形式的LPTS-L融合蛋白。采用Ni Sepharose4B柱亲和层析,可以获得纯度较高的蛋白,但不适合大量制备。通过设计引物去掉了pET-24a载体上的6×His tag将LPTS-L基因进行了非融合表达,然后采用磷酸纤维素P11阳离子交换层析纯化LPTS-L蛋白,纯度可达到55%。再经Sephadex G-100凝胶过滤,LPTS-L蛋白的纯度可达到80%。Western blot实验显示经纯化后的LPTS-L蛋白可与兔抗GST-LPTS-L的多抗发生特异性结合。采用TRAP法测定蛋白质活性,结果显示纯化得到的LPTS-L蛋白可抑制端粒酶的活性,与采用Ni Sepharose4B纯化获得的LPTS-L融合蛋白比较,其抑制效率基本一致。因此,所建立的技术可以有效地制备LPTS-L蛋白。     

Purification of multiple forms of adenosine deaminase from rabbit intestine.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.     

Use of ZetaPrep cartridge for the purification of human recombinant interleukin 1 beta

Zetaprep mass ion-exchange media represent a rapid and efficient chromatographic tool in the separation of proteins, in place of the conventional agarose or cellulose-based gels. We adopted this method, combined with classical steps, to purify to homogeneity human recombinant interleukin 1 beta (IL-1 beta) produced from E. coli and from S. cerevisiae. An anion exchanger QAE-ZetaPrep was used to achieve a rapid partial purification of both proteins. The IL-1 beta purification was completed by gel permeation chromatography on Sephadex G-50. When the protein was produced from yeast, an intermediate chromatographic step on a hydroxylapatite column was also necessary. The isolated proteins proved to be homogeneous by electrophoresis and amino acid analysis. The biological activity of IL-1 beta produced by E. coli is comparable to that of the natural protein, while the protein produced by yeast showed very low specific activity.     

Expression of human liver arginase in Escherichia coli. Purification and properties of the product.

Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine. It is abundantly present in the liver of ureotelic animals (i.e. those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high. Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA. By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein. Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E. coli cells. E. coli-expressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase. However, E. coli-expressed arginase was a monomer of Mr 35,000, whereas the purified erythrocyte arginase was trimer of Mr 105,000. They differed also in pH- and temperature-stabilities. Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35,000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures. It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins.     

马铃薯卷叶病毒的提纯

本文提出了一个应用液氮冷冻,一步提取,蔗糖垫层差速离心,Sephadex G-200柱层析以及蔗糖密度梯度离心法纯化马铃薯卷叶病毒的程序,改进后的马铃薯卷叶病毒提纯方法,使病毒产量达到1.18mg/kg酸浆组织,病毒提取物纯度比差速离心者更高,20％蔗糖垫层差速离心能够更加有效地去除宿主细胞成份,纯化病毒的OD260/280,260/240比值分别达到1.77和1.43。     

克鲁维酵母Y-85菊粉酶的纯化和性质

克鲁维酵母(Kluyveromyces sp.)Y-85产生的胞内菊粉酶(endocellular inulinase)和胞外菊粉酶(exocellular inulinase)粗酶液分别经PEG6000-磷酸盐缓冲液双水相抽提得部分纯化酶液。前者进一步用硫酸铵分级沉淀、Protein-PAK DEAE离子交换、Protein-PAK200SW凝胶过滤后得到两个菊粉酶组分EⅠ和EⅡ;后者采用DEAE-Sephacel离子交换、Sephadex G150凝胶过滤后得到菊粉酶Eexo。经Waters 650E蛋白纯化系统鉴定,三者均呈单一的对称峰;EⅠ和EⅡ达聚丙烯酰胺盘状凝胶电泳纯。EⅠ、EⅡ和Eexo的分子量分别为42kD、65kD和57kD;三者均为糖蛋白,多糖含量分别为30％、35％和25％;I/S(Inulinaseactivity/Sucrase activity)比值分别为0.086、0.078和0.072;三者均属外切菊粉酶。EⅠ、EⅡ和Eexo酶反应最适pH分别为4.6、4.5和4.6,最适温度分别为52℃、52℃和55℃;Ag^+、Hg^(2+)和PCMB对酶活性有强烈的抑制作用;三者水解菊芋粉糖液的产物均为果糖(86.5％)和葡萄糖(13.5％)。     

Translational activity of messenger ribonucleic acid isolated from unstimulated and phytohaemagglutinin-activated lymphocytes.

Purified cytoplasmic poly(A)+ RNA isolated from unstimulated pig lymphocytes has the same ability to direct translation in a range of cell-free systems as the corresponding mRNA from 20h phytohaemagglutinin-activated lymphocytes. Additional methylation of the mRNA is not required for maximum protein synthesis in the wheat-germ cell-free system. Misleading results are obtained if the mRNA preparations used are not adequately purified, and a method suitable for routine assessment of the degree of purification achieved is described. Cell-free protein-synthesizing systems from unstimulated lymphocytes translate added lymphocyte mRNA with lower efficiency than do comparable systems from phytohaemagglutinin-activated lymphocytes, whatever the source of the mRNA used.     

穴居狼蛛毒中一个抗菌活性多肽的鉴定和纯化

从我国新疆地区产穴居狼蛛的毒液中分离纯化了一种多肽——狼蛛抗菌肽(Lycosin)。穴居狼蛛的粗毒在酸性聚丙烯酰胺凝胶电泳中可分出11条蛋白带,用0.6％的大肠杆菌琼脂胶覆盖在凝胶上进行鉴定表明,电泳迁移率最快的区带有抗菌活性。经鉴定该多肽分子系由43至45个氨基酸残基组成,N-末端为丙氨酸,分子中的碱性和疏水性氨基酸分别占总氨基酸残基数的1/4和1/3。     

An easy cell-free protein synthesis system dependent on the addition of crude Escherichia coli tRNA

The protein-synthesizing S30 extract of Escherichia coli contains tRNA, which limits its applications in cell-free protein synthesis. Here, we show that at least Arg- and Ser-acceptor activities can be removed from a standard S30 extract by treatment with an immobilized RNase A resin. This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E. coli tRNA and a small amount of human placental RNase inhibitor. The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor. Chloramphenicol acetyltransferase was synthesized with this system and found to be active.     

Fish gonadotropin(s). II. Isolation of gonadotropin(s) from chum salmon pituitary glands using affinity chromatography.

A highly purified gonadotropic hormone preparation has been obtained from chum salmon (Oncorhynchus keta) pituitaries by extraction with ethanolic or aqueous buffer, affinity chromatography on Con A Sepharose and gel filtration on Sephadex G-75 superfine. A purified fraction from Sephadex G-75 averaged 448 mug NIH-LH-S18/mg glycoprotein as measured by the uptake of radiophosphate into chick testes. A total of 1.1 g of salmon gonadotropin (s) (SG)/kg fresh tissue was recovered when the isolation began with an aqueous extraction. Analytical polyacrylamide gel electrophoresis (P.A.G.E.) of the purified fraction from Sephadex G-75 displayed a single broad zone in non-dissociating conditions and two bands in 8 M urea. Polyacrylamide gel electrofocusing yielded six sharp bands with an isoelectric point range of 4.38 to 5.05, and four bands with an isoelectric point range of 4.31 to 4.95 in 8 M urea. A molecular weight of 41,000 was determined by gel filtration. A subunit molecular weight of 17,800 +/- 10% was found by P.A.G.E. in 0.1% sodium dodecyl sulphate (SDS), suggesting that native SG consists of two subunits. Purified preparations were highly stable in Tris-Cl buffers and retained their activity for several months when stored at -73 degrees C.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.