中国林蛙和中华蟾蜍皮肤抗菌肽的分离纯化及其抗菌活性

分别以中国林蛙长白山亚种Rana chensinensis changbaishansis和中华蟾蜍Bufo gargarizans的鲜皮为原料,通过酸化乙醇法提取抗菌肽粗提液,再经葡聚糖凝胶层析进一步分离纯化获得抗菌肽纯品,采用滤纸片法进行抑菌活性研究.结果 表明,经Sephadex G-50和Sephadex G-100分离纯化后获得3种多肽,中国林蛙与中华蟾蜍皮肤中的活性多肽对革兰氏阴性和革兰氏阳性细菌都具有一定的抗菌作用,其中多肽Ⅲ具有最佳的抑菌效果.抗菌肽相对含量比较的结果表明,蟾蜍皮肤中抗菌活性肽的含量较高,是理想的抗菌肽提出和纯化的源材料.     

多肽的分离纯化技术研究进展

随着各种活性多肽的发现,多肽分离纯化技术的研究和开发日益受到专家的关注。介绍多肽的理化性质与活性,讨论分离多肽的色谱、层析、电泳、膜、萃取等技术的特点和方法,并展望多肽的分离纯化前景,旨在为多肽的分离纯化提供参考。     

食物源蛋白肽铁配合物的研究进展

综述食物源蛋白多肽铁配合物的制备、分离纯化、结构分析、生物活性以及安全性研究进展。     

多肽融合标签的移除策略

多肽融合标签能够赋予目标蛋白新的特性,便于目标蛋白的定位、追踪、纯化以及结构和相互作用研究．但在很多情况下,尤其是研究蛋白质结构或分离纯化药用蛋白时,需要将多肽融合标签从融合蛋白上切除,以降低和消除多肽融合标签对目标蛋白结构和功能的影响．可用来切除多肽融合标签的方法主要有4种,即化学法、内切蛋白酶法、外切蛋白酶法以及自我剪切法．介绍和比较了每种方法的基本原理、应用以及研究进展．     

纳豆中抗氧化肽的分离纯化与活性研究

从发酵的纳豆中提取具有抗氧化活性的多肽,通过分子筛层析和反相高效液相色谱对纳豆上清液进行分离纯化,并采用电喷雾串联质谱进行结构鉴定.结果表明:纳豆发酵后的蛋白(多肽)混合物经Sephadex G-50凝胶色谱进行分离纯化后得到3个组分(F1、F2和F3),其中组分F3的抗氧化活性最强,总还原力达到(8.4±0.6)mm...     

苦荞多肽的纯化、结构与体外抗氧化活性研究CSCD

为了探究苦荞多肽粗提液抗氧化活性的主要来源,需要进一步对苦荞多肽进行分离纯化,通过结构鉴定来分析抗氧化活性与多肽结构之间的关系。该研究以苦荞功能提取物废渣为原料提取苦荞粗蛋白辅以植物乳杆菌发酵法制备苦荞多肽,采用大孔吸附树脂、葡聚糖凝胶、液相色谱对苦荞多肽粗提液进行分离纯化,并以抗氧化活性为指标,筛选抗氧化活性较强的组分,利用液相色谱-质谱联用技术鉴定抗氧化多肽结构。结果表明,纯度不同的苦荞多肽抗氧化能力也不同,经过Sephadex G-15葡聚糖凝胶分离纯化得到的T-3组分ABTS和DPPH自由基清除能力最优,清除率分别为96%、94%。纯化后苦荞多肽的抗氧化活性显著高于粗提液(P<0.05),但是抗氧化活性并没有完全与苦荞多肽纯度呈正相关,通过液相色谱进一步分离纯化T-3组分后得到的组分T-3-1,苦荞多肽纯度虽然达到了98%,但是对DPPH和ABTS抑制率不再增强,反而稍有下降,抑制率分别为89%和90%。由质谱鉴定结果得到,主要抗氧化肽的氨基酸序列为苯丙氨酸-脯氨酸-酪氨酸Phe-Pro-Tyr(FPY)和酪氨酸-亮氨酸-脯氨酸-苯丙氨酸Tyr-Leu-Pro-Phe(YLPF)。研究结果以期为苦荞多肽的进一步开发利用提供一定的理论基础。     

坛紫菜（Porphyra Haitanensis）中抑菌活性肽的制备与初步纯化

目的：提取坛紫菜中水溶性蛋白,并对其进行初步纯化和抑菌活性影响因素研究。方法：坛紫菜水溶性蛋白胃蛋白酶在37℃、pH1.8条件下酶解3h,再经超滤、Bio-Gel P-10和DEAE Sephadex A-50层析纯化步骤得到一定分子量范围的多肽混合物。采用平板打孔法和对金黄色葡萄球菌的抑制作用,跟踪测定活性多肽纯化过程及其活性影响因素。结果：SDS-PAGE测定结果表明该抗菌多肽分子量介于43.0KD~66.2KD之间。它对金黄色葡萄球菌生长的抑制作用随着温度的升高逐渐减弱,5%EDTA、5%柠檬酸、5%维生素C及25%二甲基亚砜对它的抑菌活性有协同作用,而5%维生素E则对它的抑菌活性有拮抗作用。结论：从坛子菜水溶性蛋白中初步纯化得到的分子量介于43.0KD~66.2KD的多肽,对金黄色葡萄球菌的生长有明显地抑制作用,并且它的抑菌活性受到温度,部分有机酸和维生素的影响。     

坛紫菜（Porphyra Haitanensis）中抑菌活性肽的制备与初步纯化

目的:提取坛紫菜中水溶性蛋白,并对其进行初步纯化和抑菌活性影响因素研究。方法:坛紫菜水溶性蛋白胃蛋白酶在37℃、pH1.8条件下酶解3h,再经超滤、Bio-Gel P-10和DEAE Sephadex A-50层析纯化步骤得到一定分子量范围的多肽混合物。采用平板打孔法和对金黄色葡萄球菌的抑制作用,跟踪测定活性多肽纯化过程及其活性影响因素。结果:SDS-PAGE测定结果表明该抗菌多肽分子量介于43.0KD~66.2KD之间。它对金黄色葡萄球菌生长的抑制作用随着温度的升高逐渐减弱,5%EDTA、5%柠檬酸、5%维生素C及25%二甲基亚砜对它的抑菌活性有协同作用,而5%维生素E则对它的抑菌活性有拮抗作用。结论:从坛子菜水溶性蛋白中初步纯化得到的分子量介于43.0KD~66.2KD的多肽,对金黄色葡萄球菌的生长有明显地抑制作用,并且它的抑菌活性受到温度,部分有机酸和维生素的影响。     

BACE蛋白的表达、纯化和活性测定

在大肠杆菌中表达、纯化并重新折叠以获得有活性的酸性蛋白水解酶 (BACE蛋白 )———一种与阿尔茨海默病 (AD)发病相关的蛋白水解酶。克隆BACE活性区的表达序列到原核表达载体 pET11a中 ,经E .coliBL2 1(DE3)表达 ,从包涵体中获取蛋白质 ,电泳鉴定后经梯度反向快速折叠法重新折叠 ,柱层析分离纯化 ,得到了表达的重组可溶性BACE蛋白 ;用高效液相色谱、质谱等方法检测其对人工合成多肽底物的水解作用 ;测定了BACE蛋白的酶促动力学常数。结果表明 ,得到的重组BACE蛋白具有水解人工合成小肽底物的活性。     

嗜线虫致病杆菌HB310菌株杀虫蛋白的纯化及活性鉴定

嗜线虫致病杆菌Xenorhabdus nematophila HB310是从河北省土壤中筛选出的一株昆虫病原线虫体内分离纯化获得的共生菌,该菌的发酵液对多种昆虫有较高的杀虫活性。利用85％饱和度的硫酸铵盐析分别获得胞内蛋白提取物和上清液中胞外蛋白提取物,生测结果表明这两种蛋白提取物中都含有胃毒素和血腔毒素。通过制备型非变性凝胶电泳对蛋白提取物进行分离和纯化,得到了3种有杀虫活性的毒素蛋白（毒素Ⅰ、毒素Ⅱ和毒素Ⅲ）,胞内的毒素蛋白与分泌到胞外上清液中的毒素蛋白是同种蛋白。毒素Ⅰ和毒素Ⅱ对棉铃虫初孵幼虫有明显的胃毒活性,但没有血腔毒性;毒素Ⅲ对大蜡螟幼虫有很强的血腔毒性,LD₅₀为0.18 μg/头。SDS-PAGE图谱显示毒素Ⅰ和毒素Ⅱ是由多个多肽组成的复合蛋白,而毒素Ⅲ只分离出一条多肽。毒素Ⅱ在50℃处理10 min,其杀虫活性没有显著变化;70℃处理10 min对毒素Ⅲ杀虫活性没有显著影响。     

Isolation of yeast ribosomal proteins L3 and L2 for immunological studies

We report on a rapid method for the isolation and purification of the yeast ribosomal proteins L3 and L2 using a simple instrumentation. Preparative dodecyl sulfate polyacrylamide gel electrophoresis was applied to the separation of cytoplasmatic ribosomal proteins of the large subunit from the yeast Saccharomyces cerevisiae. The polypeptides were removed from gel slices by electrophoretic elution. Subsequent analytical electrophoresis showed groups of proteins in all but two fractions. The latter were further analysed by a two-dimensional gel electrophoresis system which disclosed the purity of two polypeptides. They were identified as L3 and L2. Their molecular masses were 51.5 and 44 kDa as estimated from the gels. A possible application to the isolation of other yeast ribosomal proteins is discussed. An antiserum against the polypeptide L3 was raised in a rabbit. Applying an enzyme-linked immunosorbent assay (ELISA) we were able to determine the relative antibody concentration. Its specificity was demonstrated by immunoblotting.     

Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.     

A "tagless" strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking

Tandem affinity purification is the principal method for purifying and identifying stable protein complexes system-wide in whole cells. Although highly effective, this approach is laborious and impractical in organisms where genetic manipulation is not possible. Here, we propose a novel "tagless" strategy that combines multidimensional separation of endogenous complexes with mass spectrometric monitoring of their composition. In this procedure, putative protein complexes are identified based on the comigration of collections of polypeptides through multiple orthogonal separation steps. We present proof-of-principle evidence for the feasibility of key aspects of this strategy. A majority of Escherichia coli proteins are shown to remain in stable complexes during fractionation of a crude extract through three chromatographic steps. We also demonstrate that iTRAQ reagent-based tracking can quantify relative migration of polypeptides through chromatographic separation media. LC MALDI MS and MS/MS analysis of the iTRAQ-labeled peptides gave reliable relative quantification of 37 components of 13 known E. coli complexes: 95% of known complex components closely co-eluted and 57% were automatically grouped by a prototype computational clustering method. With further technological improvements in each step, we believe this strategy will dramatically improve the efficiency of the purification and identification of protein complexes in cells.     

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.     

A genetically encodable microtag for chemo-enzymatic derivatization and purification of recombinant proteins

Efficient separation of recombinant polypeptides from proteins of the expression host and their subsequent derivatisation with functional chemical groups is essential for the success of many biological applications. Numerous tag systems have been developed to facilitate the purification procedure but only limited progress has been made in development of generic methods for targeted modification of proteins with functional groups. In this work, we present a novel 6 amino acid long C-terminal protein tag that can be selectively modified with functionalized derivatives of farnesyl isoprenoids by protein farnesyltransferase. The reaction could be performed in complex protein mixtures without detectable unspecific labeling. We demonstrate that this modification can be used to purify the target protein by over 800-fold in a single purification step using phase partitioning. Moreover, we show that the fluorescent group could be used to monitor the interaction of the derivatized proteins with other polypeptides.     

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.     

蛋白质的反胶团萃取机制及动力学模型研究进展

反胶团萃取是近年发展起来的分离和纯化生化物质的新方法,本文介绍了反胶团萃取蛋白质技术的原理和机制、影响反胶团中蛋白质稳定性的因素,改进的蛋白质反萃取工艺,反胶团的酶动力学研究以及反胶团萃取技术的研究展望。     

The purified colicin S8 is a multimeric protein.

Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc. Colicins are commonly inducible and extracellular. Their molecular masses range from 30 to 90 kDa. Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure. In our hands, purified colicin S8 was an aggregation of extremely related polypeptides. Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa. Behavior on molecular filtration indicated a molecular weight higher than 200 kDa. Similar results were obtained when purification was carried out through FPLC. Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide. We conclude that more than one form of colicin S8 exists. The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides. Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.