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51.
鸭血清胆碱酯酶的纯化及性质研究 总被引:3,自引:0,他引:3
首次采用新技术双水相萃取方法作为鸭血清胆碱酯酶(EC.3.1.1.8 CHE) 纯化的第一步,后经 DEAE-Sephadex A50,sephadex G200 柱层析,获得电泳纯鸭血清胆碱酯酶,提纯倍数1018倍,酶活力回收43.4%,比活274.9U/mg。鸭血清胆碱酯酶性质研究表明:此酶是糖蛋白和酸性蛋白水解酶,等电点 4.2 左右,最适 pH7.5 左右;对底物碘化硫代丁酰胆碱的 Km=9.8×10-5mol/L;SDS-PAGE 电泳和聚丙烯酰胺梯度电泳表明,鸭血清胆碱酯酶以相同亚基组成的不同聚合体形式存在,亚基分子量 78000,具有完整的酶活性.不同聚合体带电状态相同. 相似文献
52.
Allyson Tucker Dilks Joanne Gilchrist Yik Lam Nathan Nicholes Brad Stanley 《Biotechnology progress》2023,39(4):e3351
No-salt flowthrough hydrophobic interaction chromatography (HIC) has been shown to effectively remove process and product-related impurities from bioprocess streams. In this publication, a panel of six antibodies has been used to demonstrate operating principles for the application of no-salt flowthrough HIC in antibody purification processes. The results indicate that no-salt flowthrough HIC provides robust aggregate clearance across operating conditions including flow rate, and variations in resin ligand density. Additionally, HMW reduction has an optimal pH range relative to the isoelectric point of each molecule and high molecular weight (HMW) reduction can be improved by altering the total protein load and/or HMW concentration to drive binding of high molecular weight species to the resin. 相似文献
53.
Yoko Ino Yutaro Yamaoka Kiho Tanaka Kei Miyakawa Mayuko Nishi Yasuyoshi Hatayama Hirokazu Kimura Yayoi Kimura Akihide Ryo 《Proteomics》2023,23(11):2200334
Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses. 相似文献
54.
A suitable method for extraction of floridoside phosphate synthase (FPS, UDP-galactose: sn-3-glycerol phosphate: 1→2′α-D-galactosyl transferase)from Porphyra perforata J. Ag. was developed. Two assay methods for enzyme activity were utilized, one measuring the amount of floridoside formed by using gas-liquid chromatography, the other measuring the sn-3-glycerol phosphate-dependent formation of UDP; both assays gave similar results. FPS is a soluble protein, and FPS activity in the extract as determined by the amount of product formed in vitro compared well with the in vivo rate of floridoside synthesis (4–7 μMmol product formed·h?1·g?1 fresh wt). The rate of product formation in vitro was linear up to 45 min and proportional to protein concentration in the assay mixture. The temperature optimum was 30–35° C. FPS was active over a range of pH values from 7.0–8.5. It was stable in concentrated solutions in the presence of 0.3 M ammonium sulfate, but activity was lost in diluted solution (protein concentration below 0.2 mg·mL?1) or below 0.2 M ion strength. The data suggest that FPS may be an oligomeric protein which occurs free in the cytoplasm or loosely bound to a membrane. It may also be a regulatory protein controlling the overall rate of synthesis of floridoside in vivo. 相似文献
55.
A D Brabban J Littlechild R Wisdom 《Journal of industrial microbiology & biotechnology》1996,16(1):8-14
Twenty five environmental isolates enriched for their ability to grow onN-acetylphenylalanine as sole carbon source were investigated for their hydrolytic action on (+)-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one). Strain CMC 3060, a mucoidal Gram-negative rod identified as a strain ofPseudomonas fluorescens, produced high levels of (+)lactamase, and was subsequently found to produce two distinct intracellular enantiomer-selective, -lactamases, one for each isomer. The (+)lactamase was produced constitutively whereas the (-lactamase was produced only in the presence of the substrate. The (+)lactamase was stable when stored as a frozen cell paste but unstable as a protein solution, losing activity during purification and storage. This enzyme was highly selective for the (+)lactam and showed no activity against a wide range of similar compounds. By use of rapid purification techniques and the inclusion of protease inhibitors and protein stabilisers, the (+)lactamase was purified to homogeneity by FPLC and found to be a monomer of molecular weight 61000 Da. 相似文献
56.
二氢叶酸还原酶结合底物的去除 总被引:1,自引:1,他引:0
分析了应用氨甲蝶呤(MTX-Agarose)亲和层析法提纯的鸡肝二氢叶酸还原酶的组成和性质.建立了用平面粒度胶等电聚焦法去除与酶紧密结合底物的方法.讨论了结合底物对酶构象研究的影响,并指出,用未完全去除结合底物的酶研究酶在变性过程构象变化会得到错误的结论. 相似文献
57.
采用HPIEC应用CM交换柱分离纯化E.coli工程菌表达的rIL-2,与空气氧化复性相比较,又有1倍以上的变性rIL-2在色谱过程中得到复性,且溶液pH影响rIL-2的复性和纯化效果:pH7.0时rIL-2复性率高于pH8.0时,但纯度明显低于pH8.0时分离的rIL-2. 相似文献
58.
用Bacillussphaericus63菌为材料,经DNA-Sepharose和CibacronBlueF3GA-Sepharose两步亲和层析,将Bsp63Ⅰ纯化到均一程度。酶比活力达61400U/mg蛋白。用凝胶过滤法测得该酶分子量为113800。该酶样品在SDS-PAGE中呈现为一条蛋白带,并测得其亚基分子量为56800。用DNS-Cl法测得该酶N-末端氨基酸为丙氨酸。上述结果表明该酶分子是由两个相同亚基组成。 相似文献
59.
经过硫酸铵30%~50%分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达53%,并制备了酶的结晶,该酶以SephadexG100柱层析及SDS-PAGE测得分子量约为81000,该酶的最适pH为7.0,最适温度为45℃,Zn2+、Mn2+、Fe2+、Cu2+、Co2+等重金属离子不同程度地抑制酶活性。 相似文献
60.
Futoshi Aranishi Kenji Hara Kiyoshi Osatomi Tadashi Ishihara 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1996,114(4):371-376
Cathepsin B was purified from the crude extract of carp (Cyprinus carpio) hepato-pancreas by the method involving ammonium sulfate fractionation and five sequential chromatographies monitored the activity with Z-Arg-Arg-MCA as a substrate, and the specific activity increased about 11,400 fold with a 2% recovery. Although the homogeneity of the purified cathepsin B was established on Native-PAGE, it migrated as two bands of 29,000 and 25,000 molecular weights by the single and heavy chains on SDS-PAGE, respectively. The monospecific antibody against the homogeneous cathepsin B was purified by the affinity chromatography on cathepsin B-Sepharose 4B, and did not immunologically react with rat cathepsin B, carp cathepsins H and L but only with carp cathepsin B by immunoelectrophoretic blot analysis. As the result of the tissue and liver distributions of cathepsin B, the remarkable immunological reactivities in the extracts of spleen, kidney and hepato-pancreas in carp and those of pacific cod, yellow fin tuna, skip jack tuna and common mackerel in pisces were detected with the anti-carp hepato-pancreas cathepsin B at molecular weight of nearby 29,000 or 25,000. 相似文献