Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A₁ rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A₂₈₀ units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.     

Production and Purification of Large Amounts of Rous Sarcoma Virus

Procedures are described for production and purification of large amounts of Rous sarcoma virus. The virus was produced by Rous sarcoma virus-transformed chicken embryo fibroblasts in roller culture which produced up to 6 mg of virus per day per liter of supernatant fluid. Various methods of concentrating virus were evaluated; pelleting yielded the best results in terms of recovery of infectious virus. Purification was achieved by means of successive velocity and equilibrium density centrifugation by using sucrose solutions made in low-salt buffer. A rapid method for the optical density measurement of virus concentration was also developed.     

Free gp70 from FeLV: enrichment from cell culture fluid by ferric oxide-agarose chromatography

A new chromatographic material based on beads of macroporous crosslinked agarose containing ferric oxide particles was used for enrichment of gp70--the envelope glycoprotein of feline leukemia virus (FeLV). Free gp70 was purified from cell culture fluid in one step with a recovery of 50 to 60% and a purification of about 60 times. The described procedure is a suitable first step for the purification of gp70 from large volumes of cell culture fluid.     

应用流行性出血热病毒抗NP和G1、G2单抗检测感染细胞和疫苗内的不同抗原成分

应用出血热病毒的抗核蛋白（ＮＰ）和糖蛋白（Ｇ１、Ｇ２）单克隆抗体的ＥＬＩＳＡ夹心法对感染细胞培养物和灭活疫苗内的ＮＰ和Ｇ１、Ｇ２抗原成分进行检测,结果表明感染Ｖｅｒｏ－Ｅ６细胞内ＮＰ和Ｇ１、Ｇ２含量均高于细胞外培养液上清,前者的抗原滴度分别为≥５１２和２５６,后者仅为６４和１６。比较三种不同疫苗内的抗原成分,显示乳鼠脑纯化疫苗内的ＮＰ抗原最高,滴度达３２０－≥６４０,而二种细胞疫苗（沙鼠肾和地鼠肾细胞）则较低,一般在２０－８０,相反二种细胞疫苗的糖蛋白滴度则高于脑疫苗（８－３２对２－８）该方法可用于疫苗生产过程中检测ＮＰ和Ｇ抗原成分。     

Method for rapid purification of class IIa bacteriocins and comparison of their activities

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared.     

Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one-year-old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.     

Purification of Large Amounts of Murine Ribonucleic Acid Tumor Viruses Produced in Roller Bottle Cultures

Murine ribonucleic acid tumor viruses and C-type virus particles are produced in relatively large quantities in roller bottle cultures. The viruses present in large volumes of culture fluids can be purified by a simple two-step procedure involving polyethylene glycol precipitation and equilibrium centrifugation in sucrose density gradients.     

登革病毒NS3全基因的扩增与克隆

登革热(ＤＦ)、登革出血热及登革休克综合征(ＤＨＦ/ＤＳＳ)是由登革病毒所致的两种不同临床类型的急性传染病,广泛流行于全球热带及亚热带地区。ＤＨＦ/ＤＳＳ以高热、出血、休克、高病死率为主要特征,近年来其发病率有迅速增加的趋势,已成为严重影响人类健康的公共卫生问题。迄今,ＤＨＦ/ＤＳＳ的发病机制仍不清楚,亦无有效的特异性预防方法[1]。登革病毒属于黄病毒科的黄病毒属,有Ⅰ、Ⅱ、Ⅲ、Ⅳ四个血清型,基因组为单股正链ＲＮＡ,全长约11ｋｂ,编码三种结构蛋白和七种非结构蛋白。基因组顺序为5′ＣＰｒＭＥＮＳ1ＮＳ2ａＮＳ2ｂＮＳ3Ｎ…     

Isoelectric precipitation and gel chromatography for purification of monoclonal IgM

A preparative scale purification procedure of monoclonal IgM from hybridoma culture supernatant with high protein content is described. The procedure consists of three steps starting with ultrafiltration followed by isoelectric precipitation and gel chromatography. Cells and debris from culture supernatant were removed by microfiltration. The clarified supernatant was concentrated 400-fold in a hollow fibre ultrafiltration apparatus (cut off 100 000 daltons). The concentrate was titrated with dilute histidine/HCl buffer close to the isoelectric point of the IgM. Precipitated proteins were harvested by centrifugation, washed and redissolved. The protein fraction containing the IgM was further purified by gel chromatography on a Sephacryl S300 column. This procedure leads to product recovery of 40% and purity of 99% related to total protein.     

Staphylococcal Hyaluronate Lyase: Purification and Characterization Studies

Staphylococcal hyaluronate lyase (hyaluronidase) derived from a pathogenic strain of staphylococcus was purified by means of salt fractionation with ammonium sulfate and gel filtration through Sephadex G-100. Most of the enzyme activity from concentrated culture supernatant fluids of staphylococci was obtained in a fraction precipitated by 90 to 100% saturation with ammonium sulfate. A small amount of enzyme was also precipitated by 80 to 90% saturation with the salt. The hyaluronidase-rich fractions did not contain other staphylococcal enzymes, such as coagulase, protease, lipase, and staphylokinase. These enzymes were present in the original concentrates. Molecular sieving chromatography of the partially purified enzyme by filtration through Sephadex G-100 resulted in a further increase in specific enzyme activity. However, more than one active peak was obtained after gel filtration, thus suggesting that there may be more than one molecular form of the enzyme. Immunodiffusion in agar gel of the chromatographically purified enzyme fraction, with immune serum from rabbits injected with concentrated staphylococcal culture supernatant fluids, indicated that there was one major antigen. A similar antigen, giving reactions of identity with the purified material, was present in the original culture supernatant fluid.     

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.     

Method for Rapid Purification of Class IIa Bacteriocins and Comparison of Their Activities

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared.     

Expression and purification of active recombinant ATM protein from transiently transfected mammalian cells

The gene mutated in the human disease ataxia telangiectasia (AT), termed ATM, encodes a large protein kinase involved in DNA repair and cell cycle control. Biochemical characterization of ATM function has been somewhat difficult because of its large size (approximately 370 kDa) and relatively low level of expression in several systems. The majority of studies have used immunoprecipitated ATM or purified ATM obtained through relatively complex procedures. Here, we describe an efficient method for the expression and purification of FLAG-epitope-tagged recombinant human ATM protein (F-ATM). This method utilizes the expression of F-ATM in transiently transfected 293T cells followed by anti-FLAG-agarose affinity chromatography. The transfection procedure has been optimized for large (225-cm(2)) culture flasks and F-ATM can be purified to near homogeneity as judged by SDS-PAGE. This procedure yields approximately 1 microg of catalytically active F-ATM protein/225-cm(2) flask that can be used for biochemical studies.     

Growth characteristics in cell culture and pathogenicity in mice of two terrestrial rabies strains indigenous to Canada

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel method for purification of staphylococcal enterotoxin A

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

A rapid and simple method for the purification of rat liver RNase inhibitor

A rapid and simple method for the purification of rat liver alkaline RNase inhibitor from a 105,000 g supernatant is reported. It involves protein precipitations by (NH₄)₂SO₄ and chromatography on carboxymethyl cellulose-RNase column. The purification procedure gives a 1020-fold increase in specific activity with a yield of 32%. This purified inhibitor can be stored for 5 weeks without any loss in activity.     

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies.

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.     

One-step affinity purification of Bacillus neutral proteases using bacitracin-silica

A purification procedure for neutral proteases from bacilli is described, in which bacitracin-silica was used as affinity medium. This enabled a one-step purification of the proteases directly from culture supernatant. Since neutral proteases are extremely sensitive towards autodigestion, conditions were chosen such, that autodigestion was largely prevented. Isopropanol appeared to be useful in both eluting the enzymes from the affinity medium, and inhibiting enzymatic activity during this step. The bacitracin-silica medium allowed high flow rates: with columns prepared for use in an FPLC system flow rates up to one column volume per minute were feasible, and still gave satisfactory results. The neutral proteases purified by this method were found to be homogeneous both by SDS-PAGE and analytical gel filtration.