毕赤酵母表达系统研究进展

毕赤酵母表达系统是目前最为成功的外源蛋白表达系统之一。该表达系统不存在原核表达系统的内毒素难以除去的问题 ,也不存在哺乳动物细胞表达系统的病毒和支原体污染等问题 ;并能够对目的蛋白进行类似高等真核生物的信号肽剪切、二硫键形成、糖基化等过程的翻译后蛋白加工。至今已有多种外源蛋白在该表达系统中成功表达。下面对毕赤酵母表达系统的特点及研究进展作一简要综述。     

枯草杆菌表达系统的研究进展

枯草杆菌由于具有非致病性、分泌蛋白能力强的特性的良好的发酵基础,是目前原核表达系统中分泌表达外源蛋白较理想的宿主。本阐述枯草杆菌基因表达的一般特点、表达载体、表达类型以及分泌表达存在的问题。     

pBV220/hIL-4优化表达及其包涵体复性研究

旨在对人白细胞介素-4(hIL-4)上、下游序列进行优化设计,提高在原核系统中的表达量,并对表达的包涵体进行复性研究提高复性率.利用BioSun的RNA二级结构预测模块辅助设计hIL-4起始密码子(AuG)上、下游的序列,使局部二级结构的自由能满足高表达要求;根据pBV220栽体和原核系统的特点优化引物序列提高hIL-4原核表达量.优化hIL-4包涵体复性条件和方法,提高复性率和生物活性.结果显示,成功构建了pBV220/hIL-4高效表达栽体,原核表达的重组人IL-4占细茵总蛋白的35%以上,明显高于优化前表达量;经过复性研究hIL-4包涵体复性率可达到15%以上,生物活性鉴定发现,纯化的hhIL-4蛋白的比活等同或高于国外同类产品.影响原核表达的条件较多,对表达序列的优化设计可以大大提高蛋白的表达量.针对人IL-4基因序列的优化设计提高了人IL-4基因的表达,复性方法的改良提高了复性率和生物活性.     

促进原核表达蛋白可溶性的研究进展

以大肠杆菌为代表的原核表达蛋白系统具有操作简单、周期短、成本低、表达量高等优点而成为获得外源表达蛋白的首选方案.但外源蛋白在原核宿主中往往以无生物活性的包涵体形式存在,这限制了原核表达系统的广泛应用.随着对蛋白折叠动力学、参与蛋白折叠的酶和分子伴侣等认识的不断深入,科学家们通过诱导条件优化、宿主细胞改造以及使用融合标签...     

膜蛋白LASS2的表达研究

利用多种原核表达系统、真核体外翻译系统和细菌/杆状病毒(Bac to Bac)的昆虫表达系统对一个具有重要生理功能的人的膜蛋白LASS2(Homo sapienslongevity assurance homologue 2 of yeastLAG1)进行表达研究。在原核表达系统中仅能够表达其羧基端胞外区片段却不能表达完整的LASS2蛋白,并制备了该片段的抗体。完整的LASS2蛋白能够在两种真核表达系统中进行表达,SDS-PAGE分析结果表明,表达产物分子量为约28kD的LASS蛋白, Western印迹分析也证实了这一结果, 并利用Ni-NTA树脂亲和层析将该蛋白纯化,纯度达到90%以上。     

枯草芽孢杆菌表达系统及其启动子研究进展

枯草芽孢杆菌作为一种革兰氏阳性细菌,由于其具有非致病性、分泌蛋白能力强的特性和良好的发酵基础及生产技术,是目前原核表达系统中表达和分泌外源蛋白的理想宿主,成为原核表达系统中的一种重要的模式菌株。而实现外源蛋白的高效表达的关键因素之一是使用强并可控制的启动子。目前,枯草芽孢杆菌中常用的启动子为组成型、诱导物诱导型、时期特异性及自诱导型。详细介绍枯草芽孢杆菌表达系统以及其常用启动子的优缺点,并对克隆新的启动子的方法做了总结,旨为完善枯草表达系统和工业生产外源蛋白奠定基础。     

高效表达可溶性重组蛋白表达载体——pHisSUMO

目的:构建含有IL-1基因的原核表达质粒,并对其原核表达情况进行检测,验 证pHisSUMO表达载体的高效可溶性表达.方法:以质粒pMD18-T-IL-1为模板,利用PCR获得I L-1基因克隆并将其与表达载体pET、pTYB、pHisSUMO连接,重组质粒经鉴定后转化到大肠杆 菌DH5α中,并检测其蛋白表达情况.结果:仅在pHisSUMO表达系统获得了IL-1融合蛋白的 高效可溶性表达.利用Ni-NTA纯化后的融合蛋白经SUMO蛋白酶Ⅰ切割,获得了纯度较高的成熟 蛋白且不残留任何氨基酸残基.结论:实验证明pHisSUMO表达系统有助于增加外源蛋白可溶性和表达量.     

重组蛋白表达系统的研究进展

利用基因重组表达外源蛋白在现代生物学技术的发展与应用中起着重要的作用。根据外源基因表达宿主不同,可以将表达系统大致分为两类:原核和真核表达系统。该文比较了常见的几种表达系统的优缺点,并探讨了在选择适合的表达系统时所需要考虑的因素如目标蛋白的产量、生物学活性、用途及其物理化学性质以及表达系统本身的成本、便利性及其安全性等,以便于选择适合的表达系统,优化提高重组蛋白产量等,进而更好地服务于科学研究。     

人源性乙酰胆碱受体α亚基胞外肽段的原核表达与血清学诊断研究

目的:利用原核系统获得重组乙酰胆碱受体(AchR)α亚基胞外肽段,通过血清学验证重组蛋白进行体外诊断可行性。方法:合成AchRα亚基胞外段全基因组核酸序列,构建pET-NusA-AchR-α融合表达载体,在原核系统内进行重组表达,以Western印迹和ELISA检测重组蛋白的抗原性和实用性。结果:原核重组Nus A-AchR-α蛋白具有强的免疫原性;ELISA检测200份临床阳性血清和100份健康人血清,阳性检出率达60%,阴性检出率为5%。结论:融合蛋白NusA-AchR-α可在原核系统内高表达,初步验证重组融合蛋白具有良好的抗原性和特异性。     

无细胞蛋白表达系统新进展及在生物制药工程中的应用

无细胞蛋白表达体系是一种以细胞抽提物为基础的体外合成蛋白质表达技术,具有遗传背景简单、反应操控简便等特点,已成为研究生物反应系统的重要技术手段。在研究人员的不断努力下,反应体系从原核扩展到真核蛋白质合成体系,而且目标蛋白表达量从毫克级提高到数克级每升,成本不断降低,反应规模可达到百公升级。近年来,无细胞蛋白表达系统在复杂蛋白、毒性蛋白和膜蛋白表达方面的优势逐渐体现,展示了其在生物制药领域的重要应用潜力。总之,无细胞技术已经成为异源蛋白质高效合成和生物制药领域中有巨大潜力的新策略。     

Wheat germ systems for cell-free protein expression

Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.     

Tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.     

Profile-based short linear protein motif discovery

ABSTRACT: BACKGROUND: Short linear protein motifs are attracting increasing attention as functionally independent sites, typically 3-10 amino acids in length that are enriched in disordered regions of proteins. Multiple methods have recently been proposed to discover over-represented motifs within a set of proteins based on simple regular expressions. Here, we extend these approaches to profile-based methods, which provide a richer motif representation. RESULTS: The profile motif discovery method MEME performed relatively poorly for motifs in disordered regions of proteins. However, when we applied evolutionary weighting to account for redundancy amongst homologous proteins, and masked out poorly conserved regions of disordered proteins, the performance of MEME is equivalent to that of regular expression methods. However, the two approaches returned different subsets within both a benchmark dataset, and a more realistic discovery dataset. CONCLUSIONS: Profile-based motif discovery methods complement regular expression based methods. Whilst profile-based methods are computationally more intensive, they are likely to discover motifs currently overlooked by regular expression methods.     

New developments in isotope labeling strategies for protein solution NMR spectroscopy

The development of novel isotope labeling strategies for proteins has facilitated the study of the structure and dynamics of these molecules. In addition, the recent emergence of alternative methods of bacterial expression for obtaining isotopically labeled proteins permits the study of new classes of important proteins by solution NMR methods.     

A new Gateway vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis

A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism from which the proteins are derived. Here we describe methods for efficiently identifying M. tuberculosis proteins that express in soluble form in Mycobacterium smegmatis. We have adapted the M. smegmatis expression vector pYUB1049 to the Gateway cloning system by the addition of att recombination recognition sequences. The resulting vector, designated pDESTsmg, is compatible with our in-house Gateway methods for E. coli expression. A target can be subcloned into pDESTsmg by a simple LR reaction using an entry clone generated for E. coli expression, removing the need to design new primers and re-clone target DNA. Proteins are expressed by culturing the M. smegmatis strain mc(2)4517 in autoinduction media supplemented with Tween 80. The media used are the same as those used for expression of proteins in E. coli, simplifying and reducing the cost of the switch to an alternative host. The methods have been applied to a set of M. tuberculosis proteins that form inclusion bodies when expressed in E. coli. We found that five of eight of these previously insoluble proteins become soluble when expressed in M. smegmatis, demonstrating that this is an efficient salvage strategy.     

A protocol for high throughput methods for the expression and purification of inner membrane proteins

The production of diffraction quality crystals for the structural determination of inner membrane proteins relies on obtaining large amounts of stable protein. Achieving this, by finding the correct parameters to successfully express and purify these proteins is often time-consuming and frustrating. The methods described here examine the most important parameters, in both expression and purification, quickly and simply. They take into account methods previously used in successful structural determinations of inner membrane proteins and collect and analyse data for use in further experiments and to investigate overall trends. These methods make use of histidine-tagged membrane proteins with a green fluorescent protein fusion but could be adapted easily for other proteins.     

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target‐related proteins, protein expression profiles of BF‐treated and control cells were compared using two quantitative proteomic methods, iTRAQ‐based and label‐free proteomic analysis. A total of 5428 proteins were identified in iTRAQ‐based analysis while 6632 proteins were identified in label‐free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20‐fold for upregulated and 0.83‐fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ‐based and label‐free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore^TM showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin‐related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF‐induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target‐related proteins and signal network of BF.     

Overexpression of membrane proteins from higher eukaryotes in yeasts

Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug–target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.     

Monitoring of gene expression by functional proteomics: response of human lung fibroblast cells to stimulation by endothelin-1.

Proteomic methods have been used to monitor changes in protein synthesis in the first 4 h following stimulation of human lung fibroblasts with endothelin-1. Using pulsed [(35)S]methionine labeling, about 70 proteins with altered protein synthesis could be detected, and the 35 proteins showing the largest changes were identified by mass spectrometry. The observed proteins included unexpected proteins such as Sox5, two isoforms of Rab14, Rab3A, translationally controlled tumor protein, and one protein of previously unknown function. There was a wide range of different kinetic behavior, and groups of functionally linked proteins such as Rab14, nucleophosmin,and cyclin-dependent kinase inhibitor 1B could be detected from similar kinetics. We propose that the functional proteomic methods are competitive with and have some advantages compared to expression profiling methods for monitoring gene expression.