高效液相色谱—电喷雾—质谱法测定蛋白质混合物

介绍高效液相谱－电喷雾－质谱（HPLC－ESI－MS）联用技术在生物大分子（蛋白）的分离和检测上所表现出来的优越性能,并利用LC－ESI－MS技术成功地分离、检测溶菌酶和牛血清蛋白混合物。     

溶菌酶及其应用

溶菌酶又称胞壁质酶。是一种无毒、无害、安全性很高的高盐基水解蛋白酶，且具有一定的保健作用。作为天然防腐剂的溶菌酶能选择性地分解微生物及植物细胞壁。而对没有细胞壁的人体细胞不会产生降解作用。因而被广泛应用于医药、食品等行业。本文从溶菌酶的组成、结构、性质出发，从食品、医药和科研几个方面详细论述了溶菌酶的应用及其发展前景。     

人溶菌酶N端与Exendin-4嵌合蛋白的基因克隆及原核表达

目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化.方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列.以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达.表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽.结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%.Western blotting检测显示重组蛋白为单一清晰条带.重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽.结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白.     

溶菌酶溶液的膜结晶工艺及影响因素

为研究溶菌酶溶液膜结晶的工艺过程和影响结晶效果的因素,采用超滤法从溶菌酶溶液中脱除溶剂,使溶液达到过饱和,在结晶助剂的作用下,制得了溶菌酶晶体.使用光学显微镜、扫描电镜和红外光谱对晶体进行了检测和分析.结果表明:采用截留分子质量为5 ku的疏水性聚醚砜超滤膜,以切向流方式进行超滤,当结晶助剂NaC l含量为5%、溶液流速为1000μm/s时,晶体粒度最大,达0.2mm,且晶体质量良好,符合X射线衍射分析的要求.     

Hofmeister序列阴阳离子对溶菌酶淀粉样纤维的影响

应用原子力显微镜(AFM)技术和傅里叶变换红外光谱(FTIR)技术对Hofmeister阴阳离子序列中9种阴离子(SO₄^2->F^->COO^->Cl^->Br^->NO₃^->I^->ClO₄^->SCN^-)和9种阳离子(NH₄⁺>K⁺>Na⁺>Cs⁺>Li⁺>Rb⁺>Mg²⁺>Ca²⁺>Ba²⁺)对溶菌酶淀粉样纤维化的影响进行了研究.通过AFM技术研究，发现Hofmeister序列中不同位置的阴离子对溶菌酶淀粉样纤维的微观形貌...     

Quantum Mechanics/Molecular Mechanics Simulations Show Saccharide Distortion is Required for Reaction in Hen Egg-White Lysozyme

Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on lysozyme show significant distortion of the bound saccharide is required to facilitate the catalytic reaction.     

Spectroscopic Analysis of Protein-Crowded Environments Using the Charge-Transfer Fluorescence Probe 8-Anilino-1-Naphthalenesulfonic Acid

The molecular behaviors of proteins under crowding conditions are crucial for understanding the protein actions in intracellular environments. Under a crowded environment, the distance between protein molecules is almost the same size as the molecular level, thus, both the excluded volume effect and short ranged soft chemical interaction on protein surface could induce the complicated influence on the protein behavior cooperatively. Recently, various kinds of analytical approaches from macroscopic to microscopic aspects have been made to evaluate the crowding effect. The method, however, has not been established to evaluate the surface specific interactions on protein surface. In this study, the analytical method to evaluate the crowding effect has been suggested by using a charge-transfer fluorescence probe, ANS. By employing the unique property of ANS attaching to charged residues on the surface of lysozyme, the crowding effect was focused, while the case was compared as a reference, in which ANS is confined in hydrophobic pockets of BSA. Consequently, the surface specific changes of fluorescence spectra were readily observed under the crowded environment, whereas the fluorescence spectra of ANS in protein inside did not change. This result suggests the fluorescence spectra of ANS binding to protein surface have the capability to estimate the crowding effect of proteins.     

Active bead-linked immunoassay on protein microarrays

Protein microarrays are becoming a powerful tool in proteome, biochemical, and clinical studies. In addition to the quality of arrayed immobilized probe molecules, sensitivity of the microarray-based assay is highly dependent on the detection technique. Here we suggest four simple techniques for rapid detection of analytes bound to protein microarrays. The techniques employ functionalized magnetic and non-magnetic beads moved to, from, or along the array surface by external forces. In contrast to other labeling techniques actively controlled physical labels: (i) make detection extremely fast to allow microarray reading in seconds; (ii) provide a low background due to active removal of weakly bound beads; and (iii) provide a highly sensitive detection, since one antigen-antibody bond is capable of holding bead immobilized on the array surface. In combination with the electrophoretically assisted active immunoassay we described recently such active reading allows to reduce total indirect immunoassay time to 7-10 min while having sensitivity in the femtomolar concentration range. High speed, sensitivity, and specificity make active bead-linked detection an ideal choice in rapid high-throughput screening and in emergency diagnostics.     

3种苯丙胺类药物与溶菌酶的作用机制及构效关系研究

在模拟人体生理条件下,采用荧光光谱法研究了3种不同结构的苯丙胺类药物(麻黄碱、伪麻黄碱和甲基麻黄碱)与溶菌酶之间的相互作用,计算了其结合常数、结合位点数和热力学参数,并探讨了3种药物对溶菌酶构象的影响.研究发现,三者可对溶菌酶内源性荧光产生强烈的猝灭作用,其猝灭过程均为静态猝灭.麻黄碱、伪麻黄碱和甲基麻黄碱与溶菌酶均形...     

Hydrophobicity Enhances the Formation of Protein-Stabilized Foams

Screening proteins for their potential use in foam applications is very laborious and time consuming. It would be beneficial if the foam properties could be predicted based on their molecular properties, but this is currently not possible. For protein-stabilized emulsions, a model was recently introduced to predict the emulsion properties from the protein molecular properties. Since the fundamental mechanisms for foam and emulsion formation are very similar, it is of interest to determine whether the link to molecular properties defined in that model is also applicable to foams. This study aims to link the exposed hydrophobicity with the foam ability and foam stability, using lysozyme variants with altered hydrophobicity, obtained from controlled heat treatment (77 °C for 0–120 min). To establish this link, the molecular characteristics, interfacial properties, and foam ability and stability (at different concentrations) were analysed. The increasing hydrophobicity resulted in an increased adsorption rate constant, and for concentrations in the protein-poor regime, the increasing hydrophobicity enhanced foam ability (i.e., interfacial area created). At higher relative exposed hydrophobicity (i.e., ~2–5 times higher than native lysozyme), the adsorption rate constant and foam ability became independent of hydrophobicity. The foam stability (i.e., foam collapse) was affected by the initial foam structure. In the protein-rich regime—with nearly identical foam structure—the hydrophobicity did not affect the foam stability. The link between exposed hydrophobicity and foam ability confirms the similarity between protein-stabilized foams and emulsions, and thereby indicates that the model proposed for emulsions can be used to predict foam properties in the future.