金属螯合亲和层析介质及应用研究的进展

金属螯合亲和层析具有螯合介质制备简单，吸附容量大，选择性及通用性较好，易于再生，成本低等优点，是分离纯化蛋白质等生物工程产品最有效的技术之一。本文综述了金属螯合亲和层析介质及最新应用方面的进展。     

壳聚糖微珠亲和层析介质的制备及其初步应用

目的制备壳聚糖(CTS)微珠亲和层析介质,并进行初步应用。方法采用多种化学方法对CTS的表面基团进行改造,使其活化。用此改性CTS进行羊抗人IgG及小牛血清纤维连接蛋白(FN)的纯化。结果此改性的CTS微珠粒径在40￣160μm之间,每克可溶胀至4ml。活化的CTS对人丙种球蛋白的吸附量为15￣25mg/gCTS,对明胶的吸附量为5.8￣6.5mg/gCTS。采用此方法纯化的羊抗人IgG及FN与Sepharose4B法纯化效果一致。结论已制备的壳聚糖微珠亲和层析介质,其性质稳定且价格较低,是一种可以广泛应用的亲和层析介质。     

应用于抗体药物捕获的耐碱亲和层析介质性能的比较

目的比较应用于抗体药物捕获的耐碱的4种蛋白A亲和层析介质和2种小分子亲和层析介质的性能,为单抗纯化工艺的选择提供参考。方法利用两种单抗纯品(mAb1和mAb2)测定4种蛋白A亲和层析介质MabSelectSuRe、POROS MabCaptureA、Absolute High Cap、TOYOPEARL AF-rProtein A-650F和2种小分子亲和层析介质Mabsorbent A2P HF、Fabsorbent F1P HF在停留时间分别为4、6、8 min时的动态载量;利用含mAb2的发酵液,从回收率和杂质去除方面比较6种亲和层析介质的纯化效果。结果在5%穿透点,各介质对mAb1和mAb2的动态载量在35~73 g/L之间。小分子亲和层析介质Mabsorbent A2P HF纯化mAb2的回收率为89%,其他5种介质纯化mAb2的回收率均≥96%;6种介质纯化mAb2的宿主细胞蛋白(host cell protein,HCP)在2 000 ppm之内,蛋白A残留量在20 ppm之内。结论结合流速、动态载量、回收率和杂质去除效果等指标,可从6种亲和层析介质中挑选适用于抗体类药物下游纯化工艺的耐碱型蛋白A或小分子亲和介质。     

亲和层析介质非特异性吸附的定量表征研究

针对亲和层析介质的非特异性吸附,基于脉冲响应法,建立了定量表征方法。以牛血清白蛋白、酵母发酵液和CHO细胞培养液作为典型杂质,考察了四种蛋白A亲和层析介质和两种基质微球,在不同pH和盐浓度条件下非特异性吸附。发现介质和基质对杂质均有不同程度的吸附,在酸性条件下非特异性吸附相对较强。对于不同料液,存在不同的杂质吸附机制,可通过静电、疏水作用或两者共同作用结合。两种介质和基质的比较结果表明,琼脂糖基介质的非特异性吸附主要依赖其基质微球与杂质间相互作用,而聚甲基丙烯酸酯基介质对杂质的吸附作用强于其基质微球。结果表明,本文建立的非特异性吸附定量表征方法切实可行,可用于量化表征介质非特异性吸附,探究相关作用机制,为介质研发提供新的分析方法和依据。     

水介质中微波促进的钯催化偶联反应研究进展

钯催化的偶联反应是形成新的C-C键的重要方法之一,一直是有机化学研究的热点和前沿领域。近年来,水介质中微波促进的钯催化偶联反应受到了合成化学家的广泛关注,一方面是由于微波加热是一种内源性加热,具有加热速度快、热转化效率高、节约能源、安全可控等优点;另一方面,水作为一种廉价易得、环境友好的反应介质代替有毒、易挥发的有机溶剂,符合现代绿色有机合成的发展方向。该综述总结了近年来水相中微波促进的钯催化偶联反应研究进展。     

多肽配基亲和吸附介质的配基密度控制规律

以谷胱甘肽为配基,琼脂糖微球为骨架,探索将谷胱甘肽通过共价键偶联到琼脂糖微球骨架上,制备可以分离谷胱甘肽S转移酶(Glutathione S-transferase, GST)及以其为标签的融合蛋白的亲和吸附介质.采用正交实验方法考察了谷胱甘肽加入量、偶联缓冲液的pH值和反应温度对亲和介质配基密度的影响.结果发现,该反应过程中的pH值对配基密度影响最大,其次为谷胱甘肽的加入量.用所制备的亲和吸附介质纯化GST(大鼠肝脏谷胱甘肽S转移酶),发现GST的吸附量随配基密度增加而增加,但GST活性却随配基密度的增加而下降,较好的干胶配基密度为260 μmol/g.大鼠肝匀浆液经过离子交换和亲和层析两个步骤,获得了电泳纯的GST,比活力为12.08 U/mg,总活性回收率为40%以上.     

用于蛋白质酶解的一体化PDMS微流控芯片

介绍了一种自制的一体化微流控芯片的制作方法。芯片由玻璃片作模具,聚二甲基硅氧烷（PDMS）为基质。该种方法制得的芯片无需封装,微通道、贮液池在聚二甲基硅氧烷单体固化过程中一体形成,用于与质谱联接萌接口也在固化过程中被固定。在芯片微通道中固定酶,从而实现蛋白质在芯片微通道中酶解,质谱检测。     

蛋白质在云母上吸附/解吸附的原子力显微镜研究

文章以牛血清白蛋白(BSA)和溶菌酶为例利用原子力显微镜(AFM)研究了蛋白质在云母表面的吸附与解吸附行为。首次利用十二烷基苯磺酸钠(SDBS)与蛋白质分子之间的相互作用实现了蛋白质从云母上的解吸附。采用轻敲模式,获得清晰的AFM图像表明:蛋白质的吸附量随蛋白质浓度及吸附时间的增加而增大;蛋白质的解吸附程度随SDBS浓度及解吸附时间的增加而增大。     

非加热变性法提高蛋白质可酶解性的方法研究

为解决生鲜肉类未经高温处理不利于人体消化吸收的问题,并保留其独特的口感与风味.通过超声法、冻融法以及反复冻融结合超声法一系列常温变性法来处理蛋白食品,提高其可酶解性,同时对变性前后蛋白质进行光谱分析和液相色谱分析以探讨其机制.结果显示:这三种方法均可提高乳清蛋白的可酶解性,实验组的样品在200～300 nm间的吸收峰数...     

CHO-K1细胞培养上清及其外泌体的蛋白质组分析

目的:确定CHO-K1细胞能否够分泌外泌体,运用液相色谱-质谱联用技术鉴定无血清培养条件下CHO-K1细胞培养上清中的蛋白质和外泌体中蛋白质的成分.方法:将CHO-K1细胞驯化至无血清培养基中,采用exoRNeasy血清/血浆试剂盒提取培养基中的外泌体,透射电镜复染法鉴定其大小和形态,质谱法鉴定标志蛋白;提取培养上清和...     

葡聚糖接枝型高载量金属螯合介质的制备与性能

对琼脂糖凝胶微球进行烯丙基活化,再接枝葡聚糖分子,考察葡聚糖分子量等因素对葡聚糖接枝过程的影响;以葡聚糖接枝琼脂糖凝胶微球为基质,制备亚氨基二乙酸型金属螯合介质,考察葡聚糖接枝过程对金属螯合介质的孔道结构、流通性能和载量等的影响. 结果表明,分子量20~500 kDa的葡聚糖都能均匀分布于琼脂糖凝胶微球内,葡聚糖接枝量随分子量增加而增大,所制的金属螯合介质形貌、粒径及其分布基本不受影响,且具有更好的流通性能,孔道结构比商品介质Ni Sepharose 6FF更丰富. 葡聚糖接枝的金属螯合介质对带组氨酸标签的乳酸脱氢酶和睫状神经营养因子的载量分别达到19和27 mg/mL,较Ni Sepharose 6FF的载量分别提高26.6%和42.0%.     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

Improving the Solubility of Artificial Ligands of Streptavidin to Enable More Practical Reversible Switching of Protein Localization in Cells

Chemical inducers that can control target‐protein localization in living cells are powerful tools to investigate dynamic biological systems. We recently reported the retention using selective hook or “RUSH” system for reversible localization change of proteins of interest by addition/washout of small‐molecule artificial ligands of streptavidin (ALiS). However, the utility of previously developed ALiS was restricted by limited solubility in water. Here, we overcame this problem by X‐ray crystal structure‐guided design of a more soluble ALiS derivative (ALiS‐3), which retains sufficient streptavidin‐binding affinity for use in the RUSH system. The ALiS‐3–streptavidin interaction was characterized in detail. ALiS‐3 is a convenient and effective tool for dynamic control of α‐mannosidase II localization between ER and Golgi in living cells.     

甲烷氯化物催化偶联合成氯乙烯单体新技术

简要介绍了几种主要的氯乙烯单体生产工艺,证明了甲烷氯化物催化偶联合成氯乙烯单体新技术的热力学可行性,并在实验室分别以一氯甲烷和二氯甲烷为原料成功合成出氯乙烯单体,最后指出了这一新技术的优势.     

The Use of Molecular Dynamics Simulation Method to Quantitatively Evaluate the Affinity between HBV Antigen T Cell Epitope Peptides and HLA-A Molecules

Chronic hepatitis B virus (HBV), a potentially life-threatening liver disease, makes people vulnerable to serious diseases such as cancer. T lymphocytes play a crucial role in clearing HBV virus, while the pathway depends on the strong binding of T cell epitope peptide and HLA. However, the experimental identification of HLA-restricted HBV antigenic peptides is extremely time-consuming. In this study, we provide a novel prediction strategy based on structure to assess the affinity between the HBV antigenic peptide and HLA molecule. We used residue scanning, peptide docking and molecular dynamics methods to obtain the molecular docking model of HBV peptide and HLA, and then adopted the MM-GBSA method to calculate the binding affinity of the HBV peptide–HLA complex. Overall, we collected 59 structures of HLA-A from Protein Data Bank, and finally obtained 352 numerical affinity results to figure out the optimal bind choice between the HLA-A molecules and 45 HBV T cell epitope peptides. The results were highly consistent with the qualitative affinity level determined by the competitive peptide binding assay, which confirmed that our affinity prediction process based on an HLA structure is accurate and also proved that the homologous modeling strategy for HLA-A molecules in this study was reliable. Hence, our work highlights an effective way by which to predict and screen for HLA-peptide binding that would improve the treatment of HBV infection.     

A Density Functional Theory Study of the Stille Cross‐Coupling via Associative Transmetalation. The Role of Ligands and Coordinating Solvents

An associative mechanism has been computationally characterized for the Stille cross‐coupling of vinyl bromide and trimethylvinylstannane catalyzed by PdL₂ (L=PMe₃, AsMe₃) with or without dimethylformamide as coordinating ligand. All the species along the catalytic cycles that start from both the cis‐ and the trans‐PdL(Y)(vinyl)Br complexes (Y=L or S; L=PMe₃, AsMe₃ or PH₃; S=DMF) have been located in the gas phase and in the presence of polar solvents. Computations support the central role of species trans‐PdL(DMF)(vinyl)Br which react by ligand dissociation and stannane coordination in the rate‐limiting transmetalation step via a puckered four‐coordinate (at palladium) transition state comprised of Pd, Br, Sn and sp² C atoms. A donating solvent may enter the catalytic cycle assisting isomerization of cis‐PdL₂(vinyl)Br to trans‐PdL(DMF)(vinyl)Br complexes via a pentacoordinate square pyramidal Pd intermediate. In keeping with experimental observations, the activation energies of the catalytic cycles with arsines as Pd ligands are lower than those with phosphines. Polytopal rearrangements from the three‐coordinate T‐shaped Pd complexes resulting from transmetalation account for the isomerization and the C C bond formation on the reductive elimination step.     

A Deeper Insight in Metal Binding to the hCtr1 N-terminus Fragment: Affinity,Speciation and Binding Mode of Binuclear Cu2+ and Mononuclear Ag+ Complex Species

Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acid sequence of the Ctr1 ectodomain Ctr1_(1-14) encompasses the characteristic Amino Terminal Cu²⁺ and Ni²⁺ binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report a combined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation of Cu²⁺ homobinuclear complexes with Ctr1_(1-14), the percentage of which is not negligible even in the presence of a small Cu²⁺ excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduce Cu²⁺ when bound to the ATCUN motif, while it reduces Cu²⁺ when bound to the His5-His6 motif involved in the formation of binuclear species. The histidine diade characterizes the second binding site and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag⁺ complexes with Ctr1_(1-14), which are assumed to mimic Cu⁺ interaction with N-terminus of Ctr1_(1-14), were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellular antibody recognizes Ctr1_(1-14) in a different way from the longer Ctr1_(1-25) that encompasses a second His and Met rich domain.     

Microbial Transglutaminase and c‐myc‐Tag: A Strong Couple for the Functionalization of Antibody‐Like Protein Scaffolds from Discovery Platforms

Antibody‐like proteins selected from discovery platforms are preferentially functionalized by site‐specific modification as this approach preserves the binding abilities and allows a side‐by‐side comparison of multiple conjugates. Here we present an enzymatic bioconjugation platform that targets the c‐myc‐tag peptide sequence (EQKLISEEDL) as a handle for the site‐specific modification of antibody‐like proteins. Microbial transglutaminase (MTGase) was exploited to form a stable isopeptide bond between the glutamine on the c‐myc‐tag and various primary‐amine‐functionalized substrates. We attached eight different functionalities to a c‐myc‐tagged antibody fragment and used these bioconjugates for downstream applications such as protein multimerization, immobilization on surfaces, fluorescence microscopy, fluorescence‐activated cell sorting, and in vivo nuclear imaging. The results demonstrate the versatility of our conjugation strategy for transforming a c‐myc‐tagged protein into any desired probe.