Identification of carboxymethyl (CM)-binding proteins derived from Lolium multiflorum pollen extract and antibody reactivity in Brazilian allergic patients

Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1 (IgE median absorbance: 1.22; IgG4 median absorbance: 0.68) and S2 (IgE median absorbance: 1.26; IgG4 median absorbance: 0.85) in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4 (IgE: r=0.9567; IgG4: r=0.9229; P<0.0001) levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy.

Effect of ion-exchange chromatography on removal of fibronectin in von Willebrand factor / 中国输血杂志

【Objective】 To study the removal effect of fibronectin(Fn) from von willebrand factor(vWF) by ion-exchange chromatography through processing human coagulation factor Ⅷ chromatographic washing products, in order to select a method that can effectively reduce Fn without compromising the activity yield. 【Methods】 In a multi-batch process development experiment, Fractogel® EMD TMAE(M) strong anion filler produced by Merck(Germany) was used to conduct chromatography to investigate vWF ristomycins titer (vWF: RCof), vWF recovery, protein content and Fn content. 【Results】 During the development of vWF pilot purification process, the content of Fn in the samples can be effectively reduced by ion-exchange chromatography, with removal rate more than 87%, titer recovery of vWF more than 80%, and no significant change in other quality indexes. 【Conclusion】 The use of ion-exchange chromatography to purify vWF can effectively reduce the content of Fn, which has positive significance for developing new product process and improving the product quality of blood products manufacturers.

Biochemical characterization with kinetic studies of melanogenic enzyme tyrosinase from white button mushroom, Agaricus bisporus

In Agaricus bisporus, color is a key determinant for marketability and consumer acceptability. However, postharvest browning has become a major concern, affecting the overall economics of the mushroom industry. In button mushrooms, the tyrosinase enzyme (E.C.1.14.18.1) is responsible for the browning reactions by catalyzing the conversion of monophenols and diphenols into quinones which polymerize to form melanin. Thus, the present study focused on the purification and characterization of tyrosinase from A. bisporus. This enzyme was purified with a final yield of 19.71% and 32.05 purification fold. The study of enzymatic activity over a temperature (5-45°C) and pH range (3-10) showed that the optimum temperature was 35°C with pH 7. The kinetic studies revealed that Km values were different for catechol (0.71 mM) and L-dopa (0.87 mM), which indicated a higher affinity of the enzyme for catechol. Inhibition studies showed that cinnamic acid is a non-competitive inhibitor while salicylic acid is a competitive inhibitor of tyrosinase. The molecular weight of the enzyme was found to be 43 kDa and different amide regions were reflected by the FTIR spectra of the enzyme. This study may provide valuable insights into the structure, biochemical properties, and inhibition of tyrosinase enzyme for controlling mushroom browning.

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)

HbA1c levels in individuals heterozygous for hemoglobin variants / Dosagem de hemoglobina glicada em heterozigotos para hemoglobinas variantes

Summary Objective: To evaluate the levels of glycated hemoglobin (HbA1c) in patients heterozygous for hemoglobin variants and compare the results of this test with those of a control group. Method: This was an experimental study based on the comparison of HbA1c tests in two different populations, with a test group represented by individuals heterozygous for hemoglobin variants (AS and AC) and a control group consisting of people with electrophoretic profile AA. The two populations were required to meet the following inclusion criteria: Normal levels of fasting glucose, hemoglobin, urea and triglycerides, bilirubin > 20 mg/dL and non-use of acetylsalicylic acid. 50 heterozygous subjects and 50 controls were evaluated between August 2013 and May 2014. The comparison of HbA1c levels between heterozygous individuals and control subjects was performed based on standard deviation, mean and G-Test. Results: The study assessed a test group and a control group, both with 39 adults and 11 children. The mean among heterozygous adults for HbA1c was 5.0%, while the control group showed a rate of 5.74%. Heterozygous children presented mean HbA1c at 5.11%, while the controls were at 5.78%. G-Test yielded p=0.93 for children and p=0.89 for adults. Conclusion: Our study evaluated HbA1c using ion exchange chromatography resins, and the patients heterozygous for hemoglobin variants showed no significant difference from the control group.

Resumo Objetivo: Avaliar os níveis de hemoglobina glicada em pacientes heterozigotos para hemoglobinas variantes e comparar os resultados deste exame com grupo controle. Método: Trata-se de um estudo experimental, baseado na comparação do exame de hemoglobina glicada de duas populações diferentes, sendo um grupo teste, representado por indivíduos heterozigóticos para hemoglobinas variantes (AS e AC) e um grupo controle, constituído por pessoas com perfil eletroforético AA. As duas populações verificadas devem obedecer ao critério de inclusão: glicemia de jejum, hemoglobina, ureia e triglicérides normais, bilirrubina > 20 mg/dL e não fazer uso de ácido acetilsalicílico. Foram avaliados 50 indivíduos heterozigotos e 50 controles no período de agosto de 2013 a maio de 2014. A comparação dos valores de hemoglobina glicada entre indivíduos heterozigóticos e controle foi realizada por meio do desvio padrão, média e teste G. Resultados: O estudo analisou um grupo teste e um grupo controle, ambos com 39 adultos e 11 crianças. A média dos adultos heterozigotos para HbA1c foi de 5,0%, o grupo controle apresentou índice de 5,7%. Já as crianças heterozigóticas obtiveram média de HbA1c de 5,11%, enquanto as normais apresentaram valores médios de 5,78%. O valor do teste G foi de p=0,9 para crianças e p=0,89 para adultos. Conclusão: Este estudo avaliou HbA1c pela metodologia de cromatografia de coluna com resinas de troca iônica, em que pacientes com heterozigoses para hemoglobinas variantes não apresentaram uma diferença significativa em relação ao grupo controle.

ISOLATION AND PURIFICATION OF IMMUNE MODULATING LACTOFERRIN FROM MONGOL BOVINE COLOSTRUM / Шинэ санаа Шинэ нээлт

@#BACKGROUND Bovine colostrums is the milk secreted by cows during the first few days after parturition. It contains many essential nutrients and bioactive components, including growth factors, immunoglobulins, lactoperoxidase, lactoferrin and cytokines ets. Lactoferrin has been reported for its multifunctional properties such as antifungal, antibacterial, antiviral antioxidant and anticancer activities. The aims of this study focused on the isolation and purification of lactoferrin from Mongolian bovine colostrums. Lactoferrin purified using HiTrap DEAE an ion exchange chromatography. Lactoferrin purification efficiency was about 60.5%. The single band of purified lactoferrin has been observed in SDS-PAGE electrophoresis. METHODS Bovine colostrum was collected at a cow farm in the Darkhan province of Mongolia. At first the cream was separated by centrifugation (10000 xg 20 min at 4oC). In order to separate the whey, the samples were precipitated with 1mol/l to pH 4.6 and centrifuged at 10000 g 20 min again. The samples of whey were stored at -18oC to the analysis. Lactoferrin was purified by HiTrap DEAE an ion exchange chromatography using 0.005 M phosphate buffer (pH 7.7) and linear gradient NaCl from 0.25M, 0.5M, 1M. During chromatography, protein in the eluents was monitored by ultraviolet absorbation at 280 nm with the instrument. Purity test done by using polyacrylamide gel electrophoresis under denaturated condition (SDS-PAGE) method by Laemmli (1970). For HPLC determination of the lactoferrin by Shimadzu Nexera X2 HPLC system with UV/ VIS detector were used. Detection was carried out at the wavelength 280 nm. Separation was performed on a chromatographic column Protein R C18 ,2.2 x 150 mm, 5 μm particle size. Linear gradient and flow rate 0.2 ml/min were used. Mobile phase a consisted of water / acetonitrile/ trifluoroacetic acid ( 95:5:0.1). The column temperature was set at 40oC and injection volume was 10 μl. Data were collected and evaluated by software Lab Solution. An external standard method for quantification analytes was used. RESULTS Purified lactoferrin in the present study had a good concentration and purification efficiency was about 60.5 %. Protein fraction from 1M NaCl gradient delivers sharp and clean peak to HPLC chromatogram that fits intensity and retention time of standard bovine lactoferrin. Ammount of lactoferrin in bovine colostrums was 0.6 mg/ml and it`s molecular weight 80 kDa as a standard sample. The retention time of lactoferrin fraction which is purified by SDS-PAGE gel electrophoresis. The peak of fraction same compared to the standard lactoferrin 5.8 minutes by HPLC analysis. CONCLUSION Ion exchange chromatography shows reliable and easy isolation of lactoferrin from Mongol bovine colostrum.

Production, purification and characterization of L-Glutaminase from Streptomyces sp. isolated from soil.

L-Glutaminase, an amidohydrolase enzyme has been a choice of interest in the treatment of lymphoblastic leukaemia. The present study reports production, purification and characterization of extracellular glutaminase enzyme from Actinomycetes. Screening was performed for twenty Actinomycetes isolates from soil; one isolate (Isolate 2) was finally selected based on the activity of glutaminase (32.5 U/ml).The isolate was identified as Streptomyces sp. Effect of physicochemical factors namely temperature, pH, NaCl concentration, and supplementary carbon & nitrogen sources on the production of L-glutaminase from the Streptomyces sp. was carried out. The enzyme production was found to be optimum with glucose as carbon source (33 U/ml), L-glutamine as nitrogen source (33.1 U/ml), at 7 pH (32.8 U/ml), temperature 30oC (32.4 U/ml) and for 0.1% NaCl concentration (32.5 U/ml). The L-glutaminase produced from Streptomyces sp. was purified by ammonium sulphate precipitation, dialysis method and ion exchange chromatography. After the purification of the enzyme by ion exchange chromatography, it has been purified 46-fold from cell-free extract and yield was 3.25%. Characterization of extracellular L-glutaminase showed that the enzyme shown optimal activity at temperature of 30°C, pH 7, at 2% NaCl and for 0.04M substrate and the Km value was calculated to be 2.8mM and Vmax was 7.57 U/ml. The molecular weight of enzyme as determined by sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) was found to be 50 kDa.

Production of Brazilian human norovirus VLPs and comparison of purification methods

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

Utilization of coconut oil mill waste as a substrate for optimized lipase production, oil biodegradation and enzyme purification studies in Staphylococcus pasteuri

Background Oil and grease laden wastewaters pose hindrance to the treatment units and further threaten the receiving water bodies. Lipase-producing microbial strains are increasingly being exploited for the remediation of such effluents. Results When bacterial strains isolated from oil mill effluent were screened for their lipolytic activity, two isolates, COM-4A and COM-6B showed significant extracellular lipase activity. They were identified to be Staphylococcus pasteuri and Bacillus subtilis, respectively. S. pasteuri COM-4A was cultivated in nutrient media based on coconut oil mill waste (CMW), in which it showed good growth at concentrations up to 20 g/L. While growing in such media, it was capable of producing lipase and other important extracellular hydrolytic enzymes. Furthermore, the isolate was able to effectively biodegrade the CMW supplemented in the medium. Applying the Box Behnken Design of Response Surface Methodology, lead to a 1.4-fold increase in both lipase production and oil removal by the isolate. The lipase was purified 9.02-fold and the molecular weight of the monomeric enzyme was deduced to be around 56 kDa. Characterization of the enzyme revealed it to be alkaliphilic and moderately thermophilic in nature, with pH and temperature optima of 9.0 and 50°C, respectively. The enzyme was also quite stable in the presence of water-miscible organic solvents. Conclusion Hence, the COM-4A lipase could be considered to be suitable for a variety of industrial applications such as in detergent formulations and in biodiesel production as well, apart from the possibility of applying it for bioremediation of fat and oil contaminants.

Comparison of glycosylated hemoglobin levels detected by 3 kinds of analytic instruments / 国际检验医学杂志

Objective To compare levels of glycosylated hemoglobin detected by 3 kinds of analytic instruments .Methods 75 samples were measured by D-10 glycosylated hemoglobin automatic analyzer(ionexchange chromatography) ,HA-8160 glycosylated hemoglobin automatic analyzer(affinity chromatography) and 7170A automatic analyzer(immune turbidimetry) .Results were tested by the homogeneity of variance ,the one-way analysis of variance and correlation analysis .Results There was a positive correlation between D-10 glycosylated hemoglobin automatic analyzer and HA-8160 glycosylated hemoglobin automatic analyzer(r2 =0 .996) , the linear equation was Y=0 .953X+0 .519 .There was a positive correlation between D-10 glycosylated hemoglobin automatic ana-lyzer and 7170A automatic analyzer(r2 =0 .996) ,the linear equation was Y=0 .925X+0 .576 .There was a positive correlation be-tween HA-8160 glycosylated hemoglobin automatic analyzer and 7170A automatic analyzer(r2 =0 .998) ,the linear equation was Y=0 .969 X+0 .081 .Conclusion In the premise of quality control in laboratory ,three different instrument can use to detect the level of glycosylated hemoglobin .

Identification and purification of major allergens in Artemisia sieversiana pollen / 中国免疫学杂志

Objective:To isolate,identify and purify the Artemisia sieversiana pollen ,the mostly widespread pollen among the Artemisia pollens in China.Methods: Artemisia sieversiana extract was precipitated by saturated ammonium sulfate and then electrophoresed by SDS-PAGE.The molecular mass of each protein band was determined by gel media system.The primary allergen proteins were identified by Western blot.Allergen proteins were purified and identified by DEAE-cellulose DE-52 ion exchange chroma-tography ( IEC) and Western blot.Results: We isolated more than twenty protein bands from Artemisia sieversiana pollen extract , including the most abundant six bands whose Mr were 62 kD,57 kD,38 kD,29 kD,25 kD,14 kD espectively.The protein bands with Mr were 62 kD and 16 kD had the highest binding capacity with the specific IgE from Artemisia pollen allergic patients.The DEAE-cellulose DE-32 IEC was used to purify the primary allergen proteins with Mr 62 kD and 16 kD.Conclusion:The primary allergens of Artemisia sieversiana include the allergen proteins whose Mr are 62 kD,16 kD and the allergen of Mr 62 kD and 16 kD can be purified by chromatography.

Evaluation on effect of ultrafiltration technology on endotoxin removal from recombinant MUC1-MBP fusion protein / 吉林大学学报(医学版)

Objective To evaluate the effectiveness of ultrafiltration technology in endotoxin removal from purified recombinant MUC1-MBP fusion protein (MUC1-MBP)and to demonstrate the effect of ultrafiltration on endotoxin removal.Methods CM Sepharose FF weak cation exchange (CM)(CM group), CM combined with Phenyl Sepharose 6 FF exchange (C6)(CM+C6 group),CM combined with ultrafiltration (CM+ultrafiltration group), and CM combined with C6 and ultrafiltration (CM+C6+ultrafiltration group)were used to purify the MUC1-MBP from E.coli. and remove endotoxin;the expression level of endotoxin was detected by Chromogenic End-point Tachypleus Amebocyte Lysate.Results There was a single band at the expected molecular weight of 62 000 by SDS-PAGE analysis.and the purity>96% by Quantity One analysis.The endotoxin levels in CM group and CM +C6 group were quite high and there was no significant difference between two groups (P>0.05 );the endotoxin level in CM+ultrafiltration group was significantly lower than that in CM group, and there was significant difference (P0.05 ). Conclusion The effects of CM or CM combined with C6 on endotoxin removal are quite poor, especially C6;CM combined with ultrafiltration are quite effective on endotoxin removal,and ultrafiltration plays an important role in endotoxin removal.

A new methodology for polyvalent intravenous immunoglobulin solution production with a two-stage process of viral inactivation

Highly purified intravenous immunoglobulin G concentrate (IV IgG) was produced with the use of polyethylene glycol associated to a single-stage precipitation by ethanol, instead of the classic Cohn-Oncley process, which employs cold alcohol as the precipitating agent, in a three-stage process. Precipitation of crude fraction containing more than 95% of immunoglobulin G was performed by liquid chromatography with a cation exchanger, CM-Sepharose, as a stationary phase. During the process, the product was subjected to two-stage viral inactivation. The first stage was performed by the action of sodium caprylate, 30 mM at pH 5.1+/- 0.1, and the second stage was performed by the action of a solvent-detergent mixture. The finished product was formulated at 5% with 10% sucralose as the stabilizing agent. The process yields 3.3g of IgG/liter of plasma. The finished product analysis showed an anti-complementary activity lower than 1CH50. Polymer and aggregate percent levels were lower than 3% in the five batches studied. The analysis of neutralizing capacity showed the presence of antibacterial and antiviral antibodies in at least three times higher concentrations than the levels found in source plasma. The finished product fulfilled all purity requirements stated in the 4th edition of the European pharmacopeia.

Obteve-se concentrado de imunoglobulina G intravenosa IgGIV, altamente purificado, utilizando-se polietilenoglicol associado a uma única etapa de precipitação por etanol, em substituição ao tradicional método descrito por Cohn-Oncley, que emprega, em três etapas, o mesmo álcool resfriado, como agente precipitante. A purificação da fração bruta contendo mais de 95% de imunoglobulina G foi realizada utilizando-se cromatografia líquida com um trocador de cátion, a CM-Sepharose, como fase estacionária. Durante o processamento o produto foi submetido a dupla inativação viral sendo a primeira pela ação do caprilato de sódio, 30 mM a pH 5,1+/- 0,1 e a segunda por ação de mistura de solvente/detergente. O produto acabado foi formulado a 5% utilizando-se sucralose 10% como estabilizante. O rendimento da metodologia foi de 3,3g de IgG/litro de plasma. A análise do produto acabado demonstrou atividade anti-complementar inferior a 1CH50. O valor percentual de polímeros e agregados em cinco lotes realizados foi inferior a 3%. O estudo da capacidade de neutralização demonstrou a presença de anticorpos anti-bacterianos e anti-virais em concentração pelo menos três vezes maior que o plasma de origem. O produto acabado apresentou conformidade com todos os requisitos de pureza dispostos na farmacopéia européia IV edição.

Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.

O propósito deste trabalho foi melhorar a separação e o rendimento das isoformas puras β- e α-tripsina por meio de cromatografia de troca iônica e caracterizar algumas propriedades físico-químicas dessas isoformas. A purificação de isoformas de tripsina foi realizada em SE Sephadex, com tampão tris-HCl, pH 7,10 a 4ºC. A quantidade de amostra, a concentração salina, o fluxo e o pH da fase móvel foram variados para determinar o efeito sobre a resolução da separação. A resolução foi otimizada principalmente entre β- e α-tripsina, utilizando o pH 7,10 a 4ºC. Isoformas puras foram obtidas a partir de 100 mg de tripsina comercial bovina depois de sete dias de cromatografia, fornecendo 51,0 mg de β-tripsina totalmente pura e 13,0 mg de α-tripsina parcialmente pura, com quantidades pequenas de contaminação por ψ-Tripsina. Assim, tempo e resolução da purificação foram otimizados redendo grandes quantidades de enzimas puras e ativas que são úteis em várias áreas de pesquisa e ciências biotecnológicas.

Optimization of Expression by Response Surface Methodology and Purification of Recombinant Human Cardiac Troponin-I(cTnI) in Escherichia coli / 中国生物工程杂志

To optimize the growth condition for the established gene engineer bacteria express cardiac troponin-I(cTnI) and to obtain purified cTnI as an antigen to produce clinical assay kits used in acute myocardial injury(AMI) diagnosis.Plackett Burman Design(PBD) was applied to select the factors which effect the expression of cTnI in Escherichial coli(E.coli) mostly.Induction time,pH and KCl were proved influenced expression of cTnI notably.Afterward,Response Surface Methodology(RSM) as second step to optimize the selected three factors,an equation was deduced to predict the percent of cTnI.In the most optimized condition,the percent of cTnI can reach to 26% of total cell protein.The procedures of purification included ammonium sulfate deposition and DEAE Cellulose ion exchange chromatography.SDS-PAGE shows that purified cTnI contain one band.cTnI could be used to immune animals as an antigen to produce monoclonal antibodies with high affinity and specificity.It maybe as calibrators to harmony the difference assays of cTnI measurement in clinical.

Processing Technology Research of Fermentation and Purification of SUMO Protease UlP1 / 中国生物工程杂志

Nowadays,small peptides are always expressed in the form of fusion protein.The expression product contains many superfluous amino acids which can affect the biological functions of small peptides even expressed by GST fusion protein expression system.SUMO protease can cut SUMO fusion protein expressed by fusion expression system without any amino acid residues left on target protein thus become a hot topic in this field.Recombinant His-UlP1/pET3c/BL21(DE3)engineering strain was constructed by genetic engineering technology and the expression conditions were optimized in shake flaks.The process of high density fermentation was explored and different purification conditions were detected by chromatography.The results showed that SUMO protease could be expressed well after inducing the engineering strain by IPTG of 1.0mmol/L at 30℃ for 6 hours.The expression level of the strain in fermentation pots could reach 24.3% analyzed by SDS-PAGE.The purity of SUMO protease was more than 98% after further purification by cation exchange chromatography.The yield was 355mg SUMO protease per liter fermentation liquid.Western blot analysis demonstrated that there were immune reactions between IlP1 and 6?His antibodies,so it has established a good foundation for large-scale industralazation in the future.

Optimization of Separating Conditions of Mouse Gut Flora by High Performance Ion Exchange Chromatography Analysis / 中国生物工程杂志

The influence of separating effect of different chromatographic conditions of mouse gut flora by high performance ion exchange chromatography analysis was studied. The optimum chromatographic conditions for separating gut bacteria were determined. The sample was applied to the chromatography column packed with Toyopearl SuperQ-650c anion resin, equilibrated with 0.02mol/L piperazin-hydrochloric acid buffer (pH 8.0), and elution salt 1mol/L NaCl, eluted with the gradient of 0-50% NaCl/ 80 min, then 50%~75% NaCl/ 25 min at the flow rate 1ml/min, and injecting volume was 1ml.Under these conditions, intestinal flora were separated into several fractions. The establishment of HPLC analysis method will lay a foundation of further research on the components of mouse gut flora and their dynamic changes.

Isolation and identification of advanced oxidation protein products / 第三军医大学学报

Objective To isolate and identify advanced oxidation protein products from human serum albumin (AOPP-HSA), expecting to search for a method of preparing highly purified and bioactive AOPP-HSA. Methods AOPP-HSA crude products were prepared in vitro by exposing HSA to HOCl. AOPP-HSA was isolated by gel chromatography and ion-exchange HPLC. Its structural features and biological activities were characterized by UV and fluorescence spectrum, SDS-PAGE, and the experiment of TNF-? release from monocytes. Results The isolated protein was purified up to 99.4% and was dityrosine-containing protein cross-linking products with molecular weight of 700?10 3. It possessed the ability of triggering the considerable release of TNF-? from monocytes. Conclusion Highly purified and bioactive AOPP-HSA can be successfully prepared by above-mentioned two-step chromatography from AOPP-HSA crude products, which builds a basis for further study of AOPP.

The purification of anti-D antibody from IgG contained anti-D by the RhD positive red blood cell / 中国免疫学杂志

Objective:To investigate the purified methods of human anti-D antibody from IgG contained anti-D. Methods:The IgG was separated by the column ion-exchange chromatography(CIEC) from the plasma in which the content of anti-D was 0.814 ?g/ml. Then the IgG preparation contained anti-D was purified by the affinity chromatography(AC) with the O group, RhD positive red blood cell (genotype CCDee). Results:The content of non anti-D IgG were reduced about 90% by the method of AC and the proportion of anti-D could be significantly increased in the final preperation. The quality of final preparation attained reqirements of national standard of biologics. Conclusion:This method is able to purify anti-D from IgG contained anti-D and offer a reference for plasma products.

Isolation and identification of neutral polysaccharide from Aloe vera var. chinensis / 中草药

Object To carry out a deeper study on the active component and the structure of Aloe vera L. var chinensis (Haw ) Berg Methods The polysaccharide was isolated from the extract of the fresh leaf pulp of A. vera var chinensis by means of DEAE Sephadex A 25 ion exchange column chromatography and identified, through HPLC, IR, TGA, 13 CNMR and elementary analysis Results Alkaline hydrolysis proved that it is a neutral, linear polymer of a partially acetylated ? 1, 4 D mannopyranose of high purity The molar ratio of D mannose to the acetyl group in the polysaccharide was 1 64∶1 Conclusion This is the first report to uncover the presence of such polysaccharide in A vera var chinensis