雨蛙皮肤分泌液中两种活性蛋白结构和功能的研究

在本研究中我们首次从雨蛙皮肤分泌液中分离得到了一种神经毒素（命名为Anntoxin）和一种干细胞自我更新支持因子（命名为AnSF）。随后，我们通过构建雨蛙皮肤cDNA 文库，利用特异引物筛选到Anntoxin 和AnSF 的cDNA 编码序列，前者的Gene Bank 登录号为FJ598043，后者还在等待分配登录号。Anntoxin 具有60 个氨基酸，是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，构建Anntoxin 的3D-NMR 溶液结构，证实Anntoxin 不同于有三对二硫键（键组合模式：1-6，2-4，3-5）的Kunitz 类型丝氨酸蛋白酶抑制剂，它只有两对二硫键（组合模式：1-4，2-3）。AnSF 具有123 个氨基酸，在C 端具有和Calmadolin 同源的两个EF 手指结构，能够支持人类胚胎干细胞（hESC）和猴神经干细胞（rNSC）的自我更新。为了进行Anntoxin 的生物活性和结构分析，我们在体外成功表达了 Anntoxin，获得了大量的重组Anntoxin（rAnntoxin）。经过生物活性分析， rAnntoxin 和天然分离到的Anntoxin 生物活性相当，都具有很强的胰蛋白酶抑制剂活性。Anntoxin 是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，和来源于芋螺（Cone Snail）的神经毒素Conkunitzin-S1，黑色眼镜蛇毒液（black cobra, Dendroaspis polylepis polylepis）的树突毒素δ-DaTX 或蛋白酶抑制剂K 分别具有32.8%和36.7%的相同序列，和鱼类（fish）来源的Stonustoxin 也有一定的同源性。利用膜片钳技术分别检测Anntoxin 对大鼠背根神经节（rat DRG）上Na+通道，K+通道，Ca2+通道的作用，结果证明Anntoxin 对河豚毒素敏感（TTX-S）的钠离子通道（Nav）有较强的抑制活性，对 K+通道，Ca2+通道作用不明显。随后我们在非洲爪蟾卵母细胞上表达几种典型和常用于测试对亚型K+通道作用的Kv1.1，Kv1.2，Kv1.3，Kv2.1 和 Kv4.2，Kv4.3，Anntoxin 对这些亚型K+通道上的K+电流都没有明显影响。我们成功构建了Anntoxin 的3D-NMR 溶液结构（NMR 号：PDB ID 2KCR， BMRB ID 16094），证实Anntoxin 具有典型的Kunitz 结构，由反向平行的 β–折叠片和α–螺旋及转角组成梨形结构。利用RT-PCR，WesternBlot 以及 ELISA 技术，发现在皮肤、脑、肝、胃和肠中都能检测Anntoxin mRNA 转录，但只在皮肤、脑、肝和胃中有蛋白表达，表达量分别为29.5、5.39、 4.80 和2.02 微克/克鲜重，可以看出Anntoxin 在皮肤中大量表达，是皮肤分泌液中非常重要的组成部分。因为皮肤是雨蛙接触外界的第一屏障，雨蛙的生存环境中存在很多潜在威胁，比如微生物、吸血昆虫、鸟类、爬行动物、哺乳动物等，所以Anntoxin 有可能是雨蛙适应环境的重要化学武器，于是我们测试了Anntoxin 对甜菜夜蛾幼虫（Laphygma exigua Hubner）、水蛇（Enhydris plumbea）、鹌鹑（Coturnix coturnix）、昆明小鼠（Kunming mice）的急性毒性，其LD50 分别为50，450，2500 和3000 微克/千克体重，说明在华西雨蛙皮肤中大量表达的Anntoxin 对几类潜在天敌确实有较强的杀灭作用。为了检测AnSF 的生物学活性，我们在体外成功表达了AnSF，获得了大量rAnSF。设计三个浓度梯度10、100 和500ng/ml，把AnSF 和hESC 共培养，发现在10~100 ng/ml 浓度时对hESC 的自我更新有支持作用；设计三个浓度梯度10、100 和500ng/ml，把AnSF 和rNSC 共培养，发现在 10ng/ml 时对rNSC 的自我更新有较强的支持作用。在超过500ng/ml 高浓度时，AnSF 对hESC 和rNSC 都有明显的细胞毒性作用，对rNSC 的毒性作用更明显。利用RT-PCR 技术，我们检测了雨蛙的皮肤、肌肉、肝脏、胰脏、胃、肠、心脏和脑，AnSF 只在皮肤中有少量表达。这表明AnSF 可能只参与雨蛙皮肤干细胞库的维持，保持皮肤内环境稳定，因为蛙类的皮肤细胞要负责产生大量活性物质参与先天免疫和抗氧化等重要的生理活动，需要经常更新，而AnSF 的存在可能保证雨蛙皮肤干细胞库容量稳定，不断分化出各种成熟的皮肤细胞来使皮肤能够得到足够和及时的更新，保证其功能的正常行使。所以AnSF 是维持华西雨蛙皮肤内环境稳定的重要物质。

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH4HF2. The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50degreesC, even 20 fmol cytochrome c could be well digested and detected.

Affinity membrane chromatography for the analysis and purification of proteins

Affinity chromatography is unique among separation methods as it is the only technique that permits the purification of proteins based on biological functions rather than individual physical or chemical properties. The high specificity of affinity chromatography is due to the strong interaction between the ligand and the proteins of interest. Membrane separation allows the processing of a large amount of sample in a relatively short time owing to its structure, which provides a system with rapid reaction kinetics. The integration of membrane and affinity chromatography provides a number of advantages over traditional affinity chromatography with porous-bead packed columns, especially with regard to time and recovery of activity. This review gives detailed descriptions of materials used as membrane substrates, preparation of basic membranes, coupling of affinity ligands to membrane supports, and categories of affinity membrane cartridges. It also summarizes the applications of cellulose/glycidyl methacrylate composite membranes for proteins separation developed in our laboratory. (C) 2001 Elsevier Science B.V. All rights reserved.

Quantitative electrochemiluminescence detection of proteins: Avidin-based sensor and tris(2,2 '-bipyridine) ruthenium(II) label

Quantitative electrochemilumineseence (ECL) detection of a model protein, bovine serum albumin (BSA) was achieved via biotin-avidin interaction using an avidin-based sensor and a well-developed ECL system of tris(2,2'-bipyridine) ruthenium(II) derivative as label and tri-n-propylamine (TPA) as coreactant. To detect the protein, avidin was linked to the glassy carbon electrode through passive adsorptions and covalent interaction with carboxylate-terminated carbon nanotubes that was used as binder to immobilize avidin onto the electrode. Then, biotinylated BSA tagged with tris(2,2'-bipyridine) ruthenium(II) label was attached to the prepared avidin surface.

Microchip Micellar Electrokinetic Chromatography Based on One Functionalized Ionic Liquid and Its Excellent Performance on Proteins Separation

Herein, one water-soluble functionalized ionic liquid (IL), 1-butyl-3-methylimidazolium dodecanesulfonate (BAS), was designed, investigated and successfully applied to microchip micellar electrokinetic chromatography (MEKC) construction. It possessed the properties of both IL and surfactant. A fairly stable pH value similar to 7.4, which was fit to pH values of general biological buffers, was nicely placed at the optimum concentration of 20 mM BAS solution. While applying BAS solution as running buffer in poly(dimethylsiloxane) (PDMS) microfluidic systems, significantly enhanced electroosmotic flow (8-fold) and resolutions between analytes were obtained than that using other supporting electrolytes or surfactants.

The immobilization of proteins on biodegradable polymer fibers via click chemistry

A facile and efficient method to immobilize bioactive proteins onto polymeric substrate was established. Testis-specific protease 50 (TSP50) was immobilized on ultrafine biodegradable polymer fibers, i.e., (1) to prepare a propargyl-containing polymer P(LA90-co-MPCIO) by introducing propargyl group into a cyclic carbonate monomer (5-methyl-5-propargyloxycarbonyl-1,3-dioxan2-one, MPC) and copolymerizing it with L-lactide; (2) to electrospin the functionalized polymer into ultrafine fibers; (3) to azidize the TSP50, and (4) to perform the click reaction between the propargyl groups on the fibers and the azido groups on the protein.

A New Method for Analysis of Disulfide-Containing Proteins by Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry

A simple and high-throughput method for the identification of disulfide-containing peptides utilizing peptide-matrix adducts is described. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups within peptide, thus allowing the observation of the peptide-matrix adduct ion [M + n + n' matrix + H](+) or [M + n + n' matrix + Na](+) (n = the number of cysteine residues, n' = 1, 2, ..., n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides. Among several matrices tested, alpha-cyano-4-hydroxycinnamic acid (CHCA, molecular mass 189 Da) and alpha-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge resulted in a mass shift of 189 Da per cysteine, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid, ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline conditions. The underlying mechanism of the reaction of the matrix addition at sulfhydryl groups is proposed, and several factors that might affect the formation of the peptide-matrix adducts were investigated.

Ionic liquids as selectors for the enhanced detection of proteins

Herein, we report an approach for protein detection enhanced by ionic liquid (IL) selectors in capillary electrophoresis (CE), with avidin as a model protein. Hydrophilic ILs were added into the running buffer of CE and acted as selectors for sample injection, enriching the positive target and excluding the negative from the capillary. When using 3% (v/v) IL selector, the detection sensitivity of avidin was improved by over one order of magnitude, while the interference from protein adsorption was effectively avoided, even in an uncoated capillary. The electrochemiluminescence method was initially used for IL-based CE with low noise that was independent of the IL concentration, making ILs almost transparent as additives in the electrophoresis buffer.

Studies on electrostatic adsorption of proteins on modified surface by real-time surface plasmon resonance technique

The elucidation of key influence factors for electrostatic adsorption is very important to control protein nonspecific adsorption on modified surfaces. In this study, real-time surface plasmon resonance technique is used to characterize the electrostatic adsorption of two proteins (mouse IgG and protein A) on carboxymethyldextran-modified surface. The results show that protein solution pH and ionic strength are key influence factors for efficient electrostatic adsorption. The influence of protein, solution pH on the amount of electrostatic adsorption depends on the type of the charge and the charge density of both protein and modified matrix on the surface. The electrostatic adsorption process involves a competition between the positively charged protein and other positively charged species in the buffer solution. A decrease of ionic strength leads to an increasing electrostatic adsorption. The kinetic adsorption constants of protein A at different pH values were also calculated and compared.

The effect of KSCN on the partition of proteins in polyethylene glycol/(NH4)(2)SO4 aqueous two-phase system

The effect of potassium thiocyanate on the partitioning of lysozyme and BSA in polyethylene glycol 2000/ammonium sulfate aqueous two-phase system has been investigated. As a result of the addition of potassium thiocyanate to the PEG/ammonium sulfate system, the PEG/mixed salts aqueous two-phase system was formed. It was found that the potassium thiocyanate could alter the pH difference between the two phases, and, thus, influence the partition coefficients of the differently charged proteins. The relationship between partition coefficient of the proteins and pH difference between two phases has been discussed. It was proposed that the pH difference between two phases could be employed as the measurement of electrostatic driving force for the partitioning of charged proteins in polyethylene glycol 2000/ammonium sulfate aqueous two-phase system.