金属螯合亲和层析纯化金属硫蛋白

将二价铜离子螯合在Chelating Sepharose Fast Flow凝胶上制成亲和层析柱,锌诱导兔肝和镉诱导小鼠肝经匀浆、乙醇处理后上柱,用pH4.0的醋酸盐缓冲液平衡,再用pH5.2不同浓度的醋酸盐缓冲液分别洗脱,可得两个金属硫蛋白(MT)洗脱峰,经确定先后为MT-2和MT-1.分离方法比传统的凝胶过滤-离子交换法简单、省时,适于实验室规模分离纯化.     

不同感染状态下EB病毒核抗原亚型表达的研究

本实验用免疫印迹法纯化的抗ＥＢＮＡ亚型抗体,结合显微荧光分光光度检测技术,检测在不同感染状态下,三种亚型ＥＢＮＡ抗原在细胞中的表达程度。结果表明,处于潜伏感染状态下的Ｒａｊｉ细胞ＥＢＮＡ－１表达量较大,经巴豆油和正丁酸诱导进入钝挫感染状态后ＥＢＮＡ－１表达减少,而ＥＢＮＡ－２的表达增强。Ｂ＿（９５－８）细胞也有相似的趋势。表明ＥＢ病毒的激活可能与不同ＥＢＮＡ亚型表达量的改变有关。     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Purification and characterization of catalase from goat (Capra capra) lung

Catalase plays a major role in the protection of tissues from toxic effects of H₂O₂ and partially reduced oxygen species. In the present study catalase was extracted and purified 330-fold from goat lung by acetone fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-200, Blue Sepharose CL-6B and Ultrogel AcA-34. The purified enzyme was almost homogeneous as judged by polyacrylamide gel electrophoresis and FPLC. The molecular weight and Stokes' radius of the purified enzyme were 339 kDa and 127±2 Å. The enzyme had 11 sulfhydryl groups and 15 tryptophan groups per mol of the enzyme. A broad pH optimum in the range 5.2 to 7.8 was obtained. Sulfhydryl group binding agents, thiol reagents and N-Bromosuccinimide inhibited the enzyme activity. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indole acetic acid, cysteine, formaldehyde and sodium azide inhibited the enzyme non-competitively with K_i values of 1.5, 1.6, 6.7, 0.55 and 0.0017 mM, respectively.     

New method for peptide purification based on selective removal of truncation peptide impurities after SPPS with orthogonal capping

Peptide purification by high-performance liquid chromatography (HPLC) is associated with high solvent consumption, relatively large effort and lack of efficient parallelization. As an alternative, many catch-and-release (c&r) purification methods have been developed over the last decades to enable the efficient parallel purification of peptides originating from solid-phase peptide synthesis (SPPS). However, with one exception, none of the c&r systems has been widely established in industry and academia until today. Herein, we present an entirely new chromatography-free purification concept for peptides synthesized on a solid support, termed reactive capping purification (RCP). The RCP method relies on the capping of truncation peptides arising from incomplete coupling of amino acids during SPPS with a reactive tag. The reactive tag contains a masked functionality that, upon liberation during cleavage from the resin, enables straightforward purification of the peptide by incubation with a resin-bound reactive moiety. In this work, two different reactive tags based on masked thiols were developed. Capping with these reactive tags during SPPS led to effective modification of truncated sequences and subsequent removal of the latter by chemoselective reaction with a maleimide-functionalized solid support. By introducing a suitable protecting group strategy, the thiol-based RCP method described here could also be successfully applied to a thiol-containing peptide. Finally, the purification of a 15-meric peptide by the RCP method was demonstrated. The developed method has low solvent consumption, has the potential for efficient parallelization, uses readily available reagents, and is experimentally simple to perform.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Considerations for operational space definition and optimization of a no-salt flowthrough hydrophobic interaction chromatography purification step

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.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

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.     

EXTRACTION,ASSAY AND SOME PROPERTIES OF FLORIDOSIDE PHOSPHATE SYNTHASE FROM PORPHYRA PERFORATA (RHODOPHYTA)1

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.