Purification of H5N1 influenza virus by different chromatography media / 中华微生物学和免疫学杂志

Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.

Expression, purification and activity verification of human norovirus NS6 protein / 中华实验和临床病毒学杂志

Objective@#To express the NS6 nonstructural protein of human norovirus (NoV) in Escherichia coli, and to detect its enzymatic activity after purification.@*Methods@#Human NoV NS6 gene was cloned into the prokaryotic expression vector pDE1 and then was transformed into E. coli BL21 for expression. NS6 protein was purified by Ni-NTA affinity chromatography and Superdex 200 pg column. The activity of NS6 protein was determined by digestion of fusion protein 15VP1-6P at 37 ℃.@*Results@#Human NoV NS6 protein was stably and highly expressed in E. coli. After purification, the expressed product reached a purity of more than 95%, and the relative molecular weight of NS6 protein was about 23×103 Da. NS6 protein could cleave the fusion protein containing rhinovirus 3C cleavage site.@*Conclusions@#The nonstructural protein NS6 of human NoV was successfully expressed in Escherichia coli, which laid a foundation for further study on the pathogenesis of human NoV.

Prokaryotic expression and purification of recombinant CARD9-MBP fusion protein / 医学研究生学报

Objective　CARD9 can activate several pathways involved in immunity, such as NF-ΚB, MAPK, etc. However the mechanism of this process has not yet been elucidated. For conducting relevant experiments in vitro, a prokaryotic expression vector of CARD9-MBP fusion protein has to been construct, and the fusion protein need to be expressed and purified.　Methods　The coding sequence of CARD9 and MBP genes were amplified by PCR and the recombinant plasmid was correctly inserted into the pET-30a(+) vector. The recombinant plasmid was transformed into E.coli DH5α competent cells and proceeded PCR identification, restriction analysis and gene sequencing. The correct recombinant plasmid was transformed into E.coli BL21(DE3) competent cells. The target protein was induced to express by IPTG under different conditions. Relative molecular weight of the target protein was detected by SDS-PAGE electrophoresis. The CARD9-MBP fusion protein was purified by MBP maltose chromatography column and gel filtration chromatography column, and identificated by MALDI-TOF mass spectrometry after MBP-tag to be removed by HRV3C enzyme.　Results　The CARD9-MBP fusion protein was successfully constructed and confirmed by PCR and restriction analysis. The result of gene sequencing was consistent with the target sequence. The SDS-PAGE electrophoresis showed that the target protein with molecular mass (MR) about 105 000 was successfully induced to express in E.coli BL21 (DE3). A quite pure CARD9-MBP fusion protein was obtained by purification of MBP maltose chromatography column. Identification by MALDI-TOF mass spectrometry demonstrated that the target protein after MBP-tag to be removed by HRV3C enzyme is CARD9 protein. In the later stage, gel filtration chromatography column was used to obtain further pure CARD9-MBP fusion protein.　Conclusion　The prokaryotic expression vector of CARD9-MBP fusion protein was successfully constructed and a large number of soluble protein expressed. The purified target protein can be obtained by purification with MBP maltose chromatography column and gel filtration chromatography column.

Preliminary investigation on diagnostic value of polyethylene glycol precipitation and gel filtration chromatography in insulin autoimmune syndrome / 中华内分泌代谢杂志

A 65-year-old male presented with recurrent palpitation and fatigue over one year. Lab tests revealed him with hyperinsulinaemic hypoglycemia. Insulin autoimmune antibody was repeatedly negative. Imaging of the pancreas seemed to be normal. Insulin-insulin autoimmune antibody complexes were detected by polyethylene glycol precipitation and gel filtration chromatography, thus the diagnosis of insulin autoimmune syndrome was suggested. By adjusting diet and administration of acarbose, all the symptoms were evidently relieved.

Preliminary investigation on diagnostic value of polyethylene glycol precipitation and gel filtration chromatography in insulin autoimmune syndrome / 中华内分泌代谢杂志

A 65-year-old male presented with recurrent palpitation and fatigue over one year. Lab tests revealed him with hyperinsulinaemic hypoglycemia. Insulin autoimmune antibody was repeatedly negative. Imaging of the pancreas seemed to be normal. Insulin-insulin autoimmune antibody complexes were detected by polyethylene glycol precipitation and gel filtration chromatography, thus the diagnosis of insulin autoimmune syndrome was suggested. By adjusting diet and administration of acarbose, all the symptoms were evidently relieved.

Production and Characterization of Bacteriocin Produced by Lactobacillus Viridescence(NICM 2167)

The present study focused on the production optimization of bacteriocin by Lactobacillus viridescence NICM 2167 followed by its purification and characterization. The bacteriocins are antimicrobial peptides produced by many Gram positive and Gram negative bacteria.The bacteriocin produced by LAB (lactic acid bacteria) received attention in recent years due to their potential application as natural preservatives in food. Bacteriocinproduced by Lactobacillus viridescence showed broad range of antimicrobial activity against food borne pathogens. Production parameters were optimized showing highest production of bacteriocinin MRS broth with pH= 7.0 incubated at 37°C for 48 h. Bacteriocin was purified in two steps involving ammonium sulphate precipitation followed by gel filtration using Sephadex G-100. Purified bacteriocin with single band on SDS-PAGE showed molecular weight of 8.3 kDa. This purified bacteriocin was stable over wide range of pH (4-10) as well as temperatures (4°C-121°C) suggesting it as a potent candidate for preservation of various foods.

Separation of human γ-tubulin ring complexes from cell lysates using gel filtration chromatography / 军事医学

Objective To separate human γ-tubulin ring complexes (γTuRC) .Methods Cell lysates prepared from 293FT cells were separated using gel filtration chromatography .Then, the eluate fractions containing γTuRC or γ-tubulin small complexes (γTuSC ) were determined by immunoblotting .Results As the constitutive components of γTuRC,γ-tubulin,γ-tubulin complex protein 2 (GCP2), GCP3 and GCP4 were eluted and enriched in the fourth fraction .The molecular mass of eluates in the fourth fraction was about 2000 ×10 3 .Following γTuRC, the constitutive components ofγTuSC including γ-tubulin, GCP2 and GCP3 were eluted and enriched in the fourteenth fraction .The molecular mass of eluates in the fourteenth fraction was about 310 ×10 3 .Unassembled free components were washed out in the eighteenth and subsequent fractions .γTuRC could be detected in the corresponding fractions by negative-PAGE separation .ConclusionγTuRC and γTuSC were successfully separated from the unassembled free components in the fourth ( 4#) and fourteenth (14#) eluted fraction, respectively.The eluates containing ofγTuRC orγTuSC can be used for microtubule assembly research.

Expression, purification and biological activities assay of human C1q and tumor necrosis factor related protein-6 / 中国药理学通报

Aim To prepare soluble human C1 q and tumor necrosis factor related protein-6 in Escherichia coli and analyze the bioactivity. Methods Recombi-nant plasmid was transformed into E. coli expression strain, and the recombinant protein Trx-hCTRP6 was expressed induced by IPTG and then purified. Results Trx-hCTRP6 was expressed efficiently and purified using Ni-NTA affinity chromatography and Superdex G-75 column. The purified Trx-hCTRP6 was shown to be active under in vivo and in vitro assay conditions. Con-clusion Active Trx-hCTRP6 is efficiently prepared from E. coli protein expression system.

Determination of the Main Content and Entrapment Efficiency in the Brucine Nanostructured Lipid Carrier by HPLC / 中国药房

OBJECTIVE:To establish a method for determining the main contents and entrapment efficiency in the bruc-ine nanostructured lipid carrier(NLC). METHODS:HPLC was adopted to determine the main content,sephadex gel filtration meth-od was employed to separate free drug in brucine NLC to determine the entrapment efficiency. The column was Dikma C18 with the mobile phase of mobile phase A(methanol)-mobile phase B [water-acetic acid-triethylamine(230∶2.4∶0.3,V/V/V)](30∶70,V/V)at the flow rate of 1 ml/min,the detection wavelength was 265 nm,volume was 10 μl and temperature was 30 ℃. RESULTS:The linear range of brucine was 4.00-80.00μg/ml(r=0.999 9);RSDs of precision,stability and reproducibility tests were≤1.67%;av-erage recoveries of content determination and sephadex gel filtration method were respectively 99.66%(RSD=0.45%,n=9) and 99.75%(RSD=1.74%,n=9);and the average entrapment efficiency was 69.92%. CONCLUSIONS:The method is simple,re-producible and efficient,and can be used for the determination of main contents and entrapment efficiency in brucine NLC.

Purification and Characterization of α-Glucan Phosphorylase Isoform Pho 2 from Spinach Leaves.

α-Glucan phosphorylase is an important enzyme of carbohydrate metabolism. In spinach leaves, it has been reported in two multiple forms viz. Pho 2 (cytosolic) and Pho 1 (plastidial). Here, we extracted and purified Pho 2 form of α-glucan phosphorylase from spinach using salting out with ammonium sulfate, desalting using Sephadex G-25 chromatography, anion exchange chromatography using DEAE-Sepharose and gel filtration chromatography using Sepharose-4-B. The purified enzyme had a specific activity, 150 units/mg protein. There was 38% recovery and 652 fold purification after final Sepharose-4B chromatography. The purified enzyme showed a single protein band on SDS sodium dodecyl sulfate polyacrylamide gel electrophoresis having molecular weight 94,000±2000 daltons. The native molecular weight is found to be 188,000±3000 daltons as determined using gel filtration chromatography over Sephadex G-200. The Pho2 exhibited optimum pH at pH 6.0 with two half pH optima at pH 5.2 and pH 7.0. The optimum temperature of Pho2 is found to be 37ºC with two half temperature optima at 30ºC and 40ºC. The Km value of the enzyme for starch and glucose-1-phosphate is found to be 116 μg/mL and 0.55 mM, respectively.

Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive.

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.

Expression,purification and biological activity assays of global human C1 q and tumor necrosis factor related protein-2 / 中国药理学通报

Aim To prepare soluble global human C1 q and tumor necrosis factor related protein-2 in Escherichia coli. Methods Recombinant expression plasmid was transformed into strain BL21-codonplus (DE3),and the recombinant protein of Trx-gH2 was expressed by IPTG induction and then purified by Ni-NTA affinity and gel filtration chromatography.Results The purified recombinant Trx-gH2 was shown to be active under in vi-vo and in vitro assay conditions.Conclusion Active recombi-nant global hCTRP2 is efficiently prepared from Escherichia coli protein expression system.

Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was 45degrees C and the highest activity was exhibited in 35 to 45degrees C. The enzyme lost their activities almost completely (95~100%) at 80 degrees C or above and as well as bellow 25degrees C.

Two-step Tandem Chromatography Purification of Anti-human CD80 Monoclonal Antibody 4E5 from Mouse Ascites / 中国生物工程杂志

A two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0,50 mmol/L Tris-HCl and satisfactory results were obtained using a 0~0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.

Serum protein-binding of radioactive triiodothyronine in the evaluation of thyroid function using sephadex gel filtration

1. A new technique for the direct measurement of T3-serum protein complex has been devised and perfected. This procedure has several distinct advantages over the standard procedures: (a) previous iodide intake (inorganic or organic)does not vitiate the result, (b) no radioactivity is introduced into the system of the patient, (c) many variables connected with red cell or resin uptake procedures are obviated, and (d) a shorter period of time is required, 3 hours, to finish the test2. A total of 160 test runs were made on 136 subjects using this new procedure. Results revealed highly significant values for the evaluation of thyroid function3. In certain situations where probably either alterations in TBP thyroxine binding capacity or changes in the proportion of T4 and T3 are operative, T8-binding gives reliable results, where PBI does not .(Summary)

The measurement of free thyroid hormone in human serum

A method for determining circulating free thyroid hormone using Sephadex gel filtration is described. It differentiates clearly the euthyroid from the hyperthyroid states. Its most useful clinical application is in the diagnosis of clinically hyperthroid states with normal protein-bound iodine and radioactive iodine uptake. (Summary)

Isolation, purification and partial characterisation of prealbumin from cerebrospinal fluid.

Prealbumin from human cerebrospinal fluid was purified using a combination of ammonium sulphate precipitation, phenol precipitation, Polyacrylamide disc gel electrophoresis and gel filtration on Sephadex G-100. The homogeneity of the purified protein was established by Polyacrylamide gel electrophoresis and Immunoelectrophoresis. On the basis of its molecular weight (55,000), amino acid composition, electrophoretic mobility and immunological cross-reactivity, the prealbumin from cerebrospinal fluid showed complete identity with serum prealbumin. The cerebrospinal fluid prealbumin levels in various neurological disorders may have a diagnostic significance.

Peanut agglutinin induced alterations in capsular and extracellular polysaccharide synthesis and ex -planta nitrogenase activity of cowpea rhizobia.

The root exudate of Arachis hypogea (groundnut) and its seed lectin peanut agglutinin were found to stimulate the synthesis of exopolysaccharide and capsular polysaccharide of the microsymbiont cowpea Rhizobium strain JLn (c). The synthesis of capsular polysaccharide was enhanced 1·5-fold and 2-fold in the presence of peanut agglutinin and root exudate, respectively. The synthesis of capsular polysaccharide was suppressed in the presence of different forms of combined nitrogen. Quantitative differences were also detected between the exopolysaccharide of cells grown in the presence and absence of root exudate. Electron microscopic examination of negatively stained lectin-treated JLn (c) cells showed an increased deposition of capsular polysaccharide surrounding the cells. Hurthermore, ex planta nitrogenase activity of JLn(c) cells in the presence of lectin was found to be enhanced by 63% in correlation with the increased synthesis of polysaccharides.

Studies on the oxidation of tannins by Aspergillus flavus.

A method for the estimation of tannin in presence of catechin, pyrogallol, protocatechuic acid and gallic acid using polyamide column chromatography was developed. Tannin added to the growing culture of Aspergillus flavus was oxidised to different extents depending on the duration of incubation. The oxidised compound was identified in the culture filtrate as a polymerised product of tannin.