龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

Generation of polyclonal antibodies against a chemically synthesized N-terminal fragment of the bacteriocin pediocin PA-1

Six mice were immunized intraperitoneally (i.p.) with a chemically synthesized 9-mer fragment (PH1) designed from the N-terminal part of the bacteriocin pediocin PA-1 and conjugated to keyhole limpet haemocyanin (KLH). After three doses of the immunogen had been administered, serum-specific antibodies were detected by a competitive direct ELISA. Myeloma cells were injected i.p. into mice in order to obtain ascites polyclonal antibodies. Although four mice developed ascites, only mouse 2 had detectable specific antibodies in the ascites fluid. The serum and ascites antibodies were specific for PH1 but they did not recognize the whole pediocin PA-1 molecule. This is the first attempt to generate antibodies against bacteriocins with a chemically synthesized oligopeptide as immunogen. This approach still remains attractive for detection, quantification, mode of action studies and purification of bacteriocins, especially those for which the purification process is difficult or inefficient at present.     

Thiophilic paramagnetic particles as a batch separation medium for the purification of antibodies from various source materials

A preparation of thiophilic agarose-based paramagnetic particles (T-Gel) has been developed with physical characteristics (particle size and particle density) that facilitate its use as a batch separation medium suitable for the large-scale purification and isolation of immunoglobulins. The medium was used to extract immunoglobulins from a wide range of starting materials, including sera, ascites fluid, tissue culture medium, and whole blood. None of these starting materials required pretreatment such as clarification by centrifugation or filtration prior to antibody extraction. The antibody purity obtained using T-Gel compared well with that obtained using protein A agarose column chromatography. Yields were approximately 30 mg of immunoglobulins per milliliter of T-Gel, and little was required in the way of specialist equipment. The method is uncomplicated and involves a roll mix extraction overnight, followed by magnetic separation to facilitate supernatant removal and subsequent washing of the particles. Elution of bound antibodies was carried out at neutral pH to yield a concentration of immunoglobulins that was approximately 7 mg/ml. The method was found to be applicable to antibody purification from the blood serum of seven different mammalian species and for all immunoglobulin classes.     

Electrophoresis of colloidal biological particles

Microscope electrophoresis was used to measure the electrophoretic mobility of polystyrene latex particles and bacterial, and mammalian tissue cells. The submicroscopic hydrophilic colloids (gelatin, serum albumin, and staphylococcal enterotoxin B) were adsorbed on latex carrier particles to determine their electrophoretic mobility and the effect of concentration, pH, electrolyte addition, and buffer ionic strength. Mobility curves as a function of pH were established for latex particles at 1 ppm concentration indicating an isoelectric point (IEP) at pH 3.6. The IEP for Escherichia coli B cells was measured at pH 2.8, Serratia marcescens at pH 2.6, Bacillus subtilis var. niger at pH 2.9, and L strain mouse fibroblast cells at pH 4.4. Using an adsorption technique, isoelectric points were measured for proteins: gelatin (acid form) at pH 9.4, serum albumin at pH 4.9, and staphylococcal enterotoxin B at pH 6.3. Procefures for examining electrophoretic characteristics of microscopic and submicroscopic biological particles are described in order to standardize procedures and to generate results applicable to an understanding of parameters influencing concentration and purification of colloidal biological particles.     

Purification of human erythropoietin.

Human erythropoietin, derived from urine of patients with aplastic anemia, has been purified to apparent homogeneity. The seven-step procedure, which included ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielded a preparation with a potency of 70,400 units/mg of protein in 21% yield. This represents a purification factor of 930. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodium dodecylsulfate at pH 7, and in the presence of Triton X-100 at pH 6. Two fractions of the same potency and molecular size, by sodium dodecyl sulfate gel electrophoresis, but differing slightly in mobility at pH 9, were obtained at the last step of fractionation. The nature of the difference between these two components is not yet understood.     

Novel heterocyclic ligands for the thiophilic purification of antibodies

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.     

Single-step purification of pepsin-derived monoclonal antibody fragments from crude murine ascitic fluids by ceramic hydroxyapatite high-performance liquid chromatography

Ceramic hydroxyapatite (CHT) high-performance liquid chromatography (HPLC) is used to purify a variety of classes of monoclonal antibodies (mAbs) from crude murine ascites fluids. We report here that this method is also applicable for simple and efficient purification of many mAb fragments that are generated by pepsin treatment of crude ascites. F(ab')(2) fragments were quantitatively generated from IgG(1) mAbs in ascitic fluids by incubation with pepsin for 6 h at pH 3.9-4.1. Under the same conditions, pepsin also cleaved unwanted ascites components, such as albumin and transferrin to very low molecular weight polypeptides. The F(ab')(2) fragments, but not the low molecular weight products, selectively bound to and were eluted from the CHT column using a linear gradient of phosphate ion concentration over 15 min. The recovery of the F(ab')(2) fragments by CHT-HPLC was >90%. This method also allowed single-step purification of mAb fragments from distinct IgG subclasses (IgG(2a) and IgG(2b)) and IgM directly from crude digested ascitic samples. This CHT-HPLC method combined with direct pepsinolysis of murine ascites is a useful strategy for rapid purification and characterization of many types of mAb fragments.     

Purification of glucose oxidase from complex fermentation medium using tandem chromatography

A fast and efficient purification method for recombinant glucose oxidase (rGOx) for flask fermentation scale (up to 2L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-alphaMF-GOx construct and expressed extracellularly in yeast Saccharomyces cerevisiae. Hydrophobic interaction (HIC)/size exclusion (SEC)-tandem chromatographic system was designed for direct purification of rGOx from a conditioned complex expression medium with minimum preceding sample preparation (only adjustments to conductivity, pH and coarse filtering). HIC on Butyl 650s (50 mM ammonium acetate pH 5.5 and 1.5 M ammonium sulphate) absorbs GOx from the medium and later it is eluted by 100% stepwise gradient with salt free buffer directly into SEC column (Sephadex 200) for desalting and final polishing separation. The electrophoretic and UV-vis spectrophotometric analyses have proven enzyme purity after purification.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.     

Nonspecific immune modulating effects of ascites fluid and hyperimmune sera in vivo

To determine whether the appearance of interferon (IFN) and the modulation of humoral responses observed following injection of irradiated tumor cells were mediated by suppressor cells, the effects of in vivo injection of I-Js specific antibodies were studied. We found that anti-I-J-containing, as well as normal ascites fluids, obtained after repeated ip injection of complete Freund's adjuvant, contain a factor which (a) induces the appearance of serum IFN, (b) enhances the response to SRBC, and (c) suppresses the response to TNP-Ficoll, when injected 1 day before antigen. This effect is not immunologically specific, is probably not caused by intact Ig, and does not appear to be mediated by T cells. Although the nature of the factor(s) responsible for the observed results has not been fully clarified, we report these findings now as a cautionary note for the interpretation of studies where in vivo injection of unfractionated ascites fluids containing monoclonal antibodies are used.     

Adsorption-desorption of recombinant hepatitis B surface antigen (r-HBsAg) from P. pastoris on a diatomaceous earth matrix: Optimization of parameters for purification

Recombinant hepatitis B surface antigen (r-HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption-elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r-HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r-HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r-HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three- and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled-up for production purposes. (c) 1993 John Wiley & Sons, Inc.     

A procedure for the rapid isolation of serum albumin using a combination of polyethyleneglycol and ethanol

The treatment of plasma (or serum) with 25% polyethyleneglycol precipitates globulins yielding electrophoretically pure albumin in the supernatant with recoveries up to 35%. Albumin was rapidly separated from polyethyleneglycol by means of ethanol precipitation. In the presence of polyethyleneglycol, ethanol treatment is able to precipitate albumin at neutral pH, however, once the polyethyleneglycol is removed, albumin shows a strict dependency of an acidic pH. This finding may be useful for general purposes of protein purification. The procedure seems to be feasable for its application to the quick isolation of albumin at moderate scale in a research laboratory.     

Detection of a proteinase inhibitor in Aspergillus nidulans

The activity of proteinases in mycelial extracts of Aspergillus nidulans increased during storage. The rate of activation increased with temperature. Three separate proteinase activities, differing in their electrophoretic mobilities on polyacrylamide gels, were readily detected at pH 6.5. Inhibitory activity, effective against all three proteinase activities, was also detected in fractions prepared from fresh mycelial extracts. The inhibitory factor(s) were heat-stable and non-dialysable. The inhibitory activity was lost during storage of mycelial extracts. It is proposed that the inhibitory factor(s) are digested by the proteinases during storage.     

Opsonic activity of ascitic fluids by a chemiluminescent method

Cirrhotic ascites are highly susceptible to spontaneous bacterial infection, whereas carcinogenic ascites are seldom infected. This difference may be explained by differences in their chemotactic, bactericidal and/or opsomic activities. We measured the chemotactic and opsonic activity of ascitic fluids from 35 alcoholic cirrhotic ascites and of 12 peritoneal carcinogenic ascites. Chemotactic activity was measured by the under-agarose technique and opsonic activity by a luminol-enhanced method. Ascitic fluids from alcoholic cirrhosis had low chemotactic (62 ± 24.5% that of N-formylated peptide) and opsonic (67 ± 50% of normal serum) activities on normal human neutrophils. In contrast, ascitic fluids from peritoneal carcinoma were found to possess high opsonic activity (114 ± 49% of normal serum) and chemotactic activity similar to that of N-formylated peptide. During a 3-month follow-up, 11 spontaneous bacterial infections were observed among the first group against none in the carcinogenic group.     

Primate (Macaca fascicularis) transferrin: isolation and partial characterization

The serum transferrin from the primate, Macaca fascicularis is isolated by a purification protocol consisting of ammonium sulphate precipitation and column chromatography. The hexose (galactose + mannose) content of Macaca transferrin is 4.7 mole per mole of protein. Quantitative determination of the sialic acid content shows that there are two sialic acid residues per molecule of Macaca transferrin. This conclusion is supported by the neuraminidase treatment of Macaca transferrin, in which there is a 2-step decrease in electrophoretic mobility. Monoferric Macaca transferrins with Fe3+ selectively labelled at the C- and N-terminal sites (TfFec and FeNTf) are prepared at pH 5.5 and 8.5 using ferric dinitrilotriacetate [Fe(NTA)2] chelate and ferrous ammonium sulphate, respectively.     

Purification and characterization of a Z-pro-prolinal-insensitive Z-Gly-Pro-7-amino-4-methyl coumarin-hydrolyzing peptidase from bovine serum--a new proline-specific peptidase.

The study of a new proline-specific peptidase from bovine serum is presented. The enzyme readily cleaves the prolyl oligopeptidase (PO) substrate Z-Gly-Pro-MCA, liberating the fluorophore MCA, thus allowing quantification of enzyme activity. Unlike PO, however, this peptidase is completely insensitive to the PO-specific inhibitor Z-Pro-prolinal and has been designated Z-Pro-prolinal-insensitive Z-Gly-Pro-MCA-hydrolyzing peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl Sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl Sepharose, calcium phosphate cellulose and DEAE anion-exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023. SDS-PAGE and size-exclusion chromatography indicated a dimeric structure with a relative molecular mass of 174 kDa. The enzyme was stable over the pH range 2.5-10.0. Optimal activity was detected in the pH range 7.4-8.0. Isoelectric focusing revealed a pI of 5.68. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site. alpha(2)M failed to inhibit activity, suggesting oligopeptidase specificity. HPLC analysis revealed a broad substrate specificity for proline-containing peptides. Kinetic analysis indicated that ZIP had a high affinity for Z-Gly-Pro-MCA with a K(m) of 54 microM deduced. Bovine serum ZIP exhibits biophysical characteristics both similar to and different from those of PO isolated from a number of sources and may serve an important physiological function in the degradation of bioactive oligopeptides.     

Optimisation of aqueous two-phase extraction of human antibodies

The purification of human antibodies in an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) 6000 and phosphate was optimised by surface response methodology. A central composite design was used to evaluate the influence of phosphate, PEG and NaCl concentration and of the pH on the purity and extraction yield of IgG from a simulated serum medium. The conditions that maximise the partition of IgG into the upper phase were determined to be high concentrations of NaCl and PEG, low concentrations of phosphate and low pH values. An ATPS composed of 12% PEG, 10% phosphate, 15% NaCl at pH 6 was further used to purify human monoclonal antibodies from a Chinese Hamster Ovary (CHO) concentrated cell culture supernatant with a recovery yield of 88% in the upper PEG-rich phase and a purification factor of 4.3. This ATPS was also successfully used to purify antibodies from a hybridoma cell culture supernatant with a recovery yield of 90% and a purification factor of 4.1.     

小鼠腹水型肝癌H22a细胞浆胞内磷蛋白磷酸酶的纯化和性质

 磷蛋白磷酸酶是磷酸化/脱磷酸化作用中重要的调节酶。本文建立了小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶(PrP)的纯化方法。用~(32)P-酪蛋白为底物测定活力。经纯化的酶纯度提高1380倍,聚丙烯酰胺梯度凝胶电泳检查,只有一条泳带。用凝胶过滤法和聚丙烯酰胺梯度凝胶电泳法测得该酶分子量为200,000。该酶催化~(32)P-酪蛋白脱磷酸化反应的最适pH7.2,对热不稳定。