大肠杆菌Ⅰ型分泌表达系统研究进展及提高蛋白表达量的策略

大肠杆菌是表达重组蛋白最常用的宿主之一。利用大肠杆菌分泌途径胞外表达重组蛋白具有可促进蛋白正确折叠,有效减少包涵体形成,简化纯化工序等诸多优势,近年来备受关注。其中,大肠杆菌Ⅰ型分泌途径具有分泌表达速度快,蛋白活性高,对宿主代谢无影响等特点,是目前应用最广泛的分泌途径之一。综述了大肠杆菌Ⅰ型分泌系统的元件组成和分泌机理及提高Ⅰ型分泌系统蛋白表达量的有效策略,为重组蛋白生产应用提供了理论依据。     

提高大肠杆菌分泌表达重组蛋白的研究进展

大肠杆菌是表达重组蛋白的常见宿主之一。重组蛋白分泌到周质空间或胞外培养基中较之在胞内以包含体形式表达有许多优势。主要讨论大肠杆菌Ⅰ、Ⅱ型分泌机制,并总结近年来在提高重组蛋白分泌表达的策略方面取得的进展。     

基于新月柄杆菌RsaA外运机制的EspA及EspA-IL-24融合蛋白胞外分泌表达研究

目的：实现大肠杆菌分泌蛋白（Esp）A及EspA与白细胞介素（IL）-24融合蛋白的胞外分泌表达,进一步验证基于新月柄杆菌RsaA外运机制的原核胞外分泌表达载体系统的有效性和通用性,并改造优化该系统。方法：利用分子克隆手段,按RsaA分泌系统操纵子组织方式,将获得的RsaA系统元件编码序列和异源调控序列克隆至pQE30骨架质粒,构建新的胞外分泌表达质粒pQABP2S;以大肠杆菌为宿主菌诱导表达EspA及EspA-IL-24融合蛋白,并通过Westernblot检测目标蛋白在培养上清中的表达。结果：获得了新的胞外分泌表达载体pQABP2S;与对照相比,该载体宿主系统培养上清中目标蛋白EspA及EspA-IL-24的表达量明显增加。结论：在大肠杆菌中通过RsaA分泌系统可实现分子大小不同的EspA及EspA-IL-24融合蛋白的特异性分泌表达,进一步证实该分泌表达策略的有效性和通用性;调整调控序列以优化分泌系统的尝试,为此类基因工程技术平台的开发提供了借鉴。     

重组蛋白在大肠杆菌周间腔的分泌表达

大肠杆菌是用于生产重组蛋白的重要工程宿主菌。但是,要获得足够的正确折叠的蛋白还存在一定的缺陷,其中一种解决此问题的方法就是使重组蛋白分泌到大肠杆菌的周间腔里。在这篇综述中,主要讨论了使重组蛋白分泌表达至大肠杆菌周间腔的近期的研究进展。     

芽胞杆菌中重组蛋白分泌表达的优化策略及研究进展

芽胞杆菌属具有良好的蛋白表达和分泌能力,在工业酶的生产中被广泛应用,是理想的工业宿主菌,但实现蛋白分泌表达的普遍高效性还存在许多瓶颈。本文综述了芽胞杆菌的蛋白分泌表达策略,从启动子、信号肽、分泌途径、宿主和培养条件这5个方面总结了提高芽胞杆菌中分泌表达重组蛋白的方法,对芽胞杆菌高效生产工业酶有一定的参考价值,最后展望了优化芽胞杆菌分泌表达的研究方向,各种新型生物技术的发展必将推进芽胞杆菌在分泌表达领域有更深入的应用。     

基因工程重组蛋白胞外分泌Ⅰ型系统的研究进展

在基因工程领域,重组蛋白表达后定位于胞内或周质间隙,影响了其表达水平与可溶性,进而影响活性,降低了利用效率。分泌表达至胞外培养基则能较好地解决这一难题。利用细菌天然分泌系统作为重组蛋白胞外递送工具已日益成为生物工程及相关领域的常用策略,其中Ⅰ型分泌系统是研究最多、最具应用前景的。本文简要综述典型的大肠杆菌α-溶血素(HlyA)分泌系统以及更具优势的新月柄杆菌SLP(RsaA)分泌系统的生化特性、遗传特征及移植利用等方面的研究进展。     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

一种高效、稳定的分泌型原核表达载体的构建及应用

以本室构建的原核表达载体pTO-T7为基础载体,PCR合成ompT引导序列,插入该载体多克隆位点上游,构建了分泌型原核表达载体pTO—OT。将2个外源基因克隆至pTO—OT,2个重组质粒在大肠杆菌中均得以高效表达,表达量为25％～30％。Western印迹分析证实了重组蛋白在大肠杆菌中表达后可被信号肽酶有效识别,切割后的重组蛋白具有良好的免疫学活性。对重组表达菌株的连续传代实验证实了该表达载体具有良好的遗传稳定性,显示了该原核表达载体在基因工程中的应用价值。     

大肠杆菌α-溶血素分泌途径研究进展

随着生物技术的不断发展,大肠杆菌胞外分泌分子机理日渐明晰,大肠杆菌表达系统成为实现规模化制备胞外重组蛋白的途径之一。本文综述了大肠杆菌α-溶血素分泌途径的转运机制及应用前景。     

细菌Ⅶ型分泌系统的研究进展

细菌分泌系统参与细菌物质转运,是细菌蛋白或DNA胞外分泌的重要途径,与细菌的生长和致病性密切相关。迄今为止,已发现了Ⅰ～Ⅶ型分泌系统。Ⅰ～Ⅵ型分泌系统存在于革兰阴性菌中,其中Ⅳ型也存在于革兰阳性菌中;Ⅶ型则存在于革兰阳性菌中。Ⅶ型分泌系统是近年来发现的一种特殊分泌系统,能介导病原微生物毒力蛋白分泌,与宿主相互作用,并参与细菌体内锌铁平衡等,在革兰阳性菌的生长代谢及致病过程中发挥重要作用。本文综述细菌Ⅶ型分泌系统的类型、功能及表达调控,以增进对这一新型细菌蛋白分泌机制的认识。     

Secretion of recombinant proteins from E. coli

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.     

Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems

Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.     

Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters

Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.     

FlgM as a Secretion Moiety for the Development of an Inducible Type III Secretion System

Regulation and assembly of the flagellar type III secretion system is one of the most investigated and best understood regulational cascades in molecular biology. Depending on the host organism, flagellar morphogenesis requires the interplay of more than 50 genes. Direct secretion of heterologous proteins to the supernatant is appealing due to protection against cellular proteases and simplified downstream processing. As Escherichia coli currently remains the predominant host organism used for recombinant prokaryotic protein expression, the generation of a strain that exhibits inducible flagellar secretion would be highly desirable for biotechnological applications.Here, we report the first engineered Escherichia coli mutant strain featuring flagellar morphogenesis upon addition of an external inducer. Using FlgM as a sensor for direct secretion in combination with this novel strain may represent a potent tool for significant improvements in future engineering of an inducible type III secretion for heterologous proteins.     

High-level secretion of Pseudomonas fluorescens type I secretion system-dependent lipase in Serratia marcescens

The type I secretion system-dependent lipase, TliA, of Pseudomonas fluorescens was successfully produced in quantity in Serratia marcescens by coexpressing its cognate ABC transporter, TliDEF. Compared with P. fluorescens and Escherichia coli, S. marcescens showed an outstanding capacity for the secretory production of TliA, which was done with the expression vectors available for use in E. coli, and no growth phase-dependency, which was unlike the typical feature of TOSS-mediated protein secretion. Among the S. marcescens tested, the highest amount of TliA (approximately 2600 units ml(-1)) was achieved by S. marcescens KCTC 2798 containing the expression plasmid pTliDEFA-223. Our results also suggest that strains of Serratia will provide a valuable opportunity for producing other extracellular TOSS-dependent proteins effectively as well as the TliDEF-dependent TliA in this study.     

Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus

Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model. In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal. We report here the development of small, high-copy-number plasmid vectors and methods for producing an obligate expression host. The vectors are based on a narrow-host-range colE1-replicon-based plasmid commonly used in Escherichia coli, to which was added the replication origin of the IncQ plasmid RSF1010. C. crescentus strains were modified to enable plasmid replication by introduction of the RSF1010 repBAC genes at the recA locus. The small (4.0-4.5 kb) plasmids were in high copy numbers in both C. crescentus and E. coli and amenable to rapid methods for plasmid isolation and DNA sequencing. The method for introducing repBAC is suitable for other C. crescentus strains or any bacterium with an adequately homologous recA gene. Application of the vector for protein expression, based on the type I secretion system of the S-layer protein, when compared to constructs in broad-host-range plasmids, resulted in reduced time and steps required from clone construction to recombinant protein recovery and increased protein yield.     

Advanced genetic strategies for recombinant protein expression in Escherichia coli

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology. Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E. coli cytoplasm. Here we review the current most important strategies for recombinant expression in E. coli. Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology. The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.     

Translocation of green fluorescent protein by comparative analysis with multiple signal peptides

Type I and II secretory pathways are used for the translocation of recombinant proteins from the cytoplasm of Escherichia coli. The purpose of this study was to evaluate four signal peptides (HlyA, TorA, GeneIII, and PelB), representing the most common secretion pathways in E. coli, for their ability to target green fluorescent protein (GFP) for membrane translocation. Signal peptide-GFP genetic fusions were designed in accordance with BioFusion standards (BBF RFC 10, BBF RFC 23). The HlyA signal peptide targeted GFP for secretion to the extracellular media via the type I secretory pathway, whereas TAT-dependent signal peptide TorA and Sec-dependent signal peptide GeneIII exported GFP to the periplasm. The PelB signal peptide was inefficient in translocating GFP. The use of biological technical standards simplified the design and construction of functional signal peptide-recombinant protein genetic devices for type I and II secretion in E. coli. The utility of the standardized parts model is further illustrated as constructed biological parts are available for direct application to other studies on recombinant protein translocation.     

Bacterial expression and enzymatic activity analysis of ME1, a ribosome-inactivating protein from Mirabilis expansa

Ribosome-inactivating proteins (RIPs) are toxic proteins synthesized by many plants and some bacteria, that specifically depurinate the 28S RNA and thus interrupt protein translation. RIPs hold broad interest because of their potential use as plant defense factors against pathogens. However, study of the activity of type I RIPs has been hampered since their expression in Escherichia coli has typically been toxic to the model system. Mirabilis expansa, an Andean root crop, produces a type I RIP called ME1 in large quantities in its storage roots. In this study, the cDNA sequence of ME1 was used to successfully express the recombinant ME1 protein in E. coli. The production of recombinant ME1 in E. coli was confirmed by Western blot analysis using anti-ME1 antibodies. The studies with fluorescence-labeled ME1 showed that ME1 can enter bacteria and be distributed in the cytoplasm uniformly, indicating its ability to access the protein synthesis machinery of the bacteria. The recombinant enzyme was active and depurinated yeast ribosomes. However, both native and recombinant ME1 proteins failed to depurinate the E. coli ribosomes, explaining the non-toxicity of recombinant ME1 to E. coli. Structural modeling of ME1 showed that it has folding patterns similar to other RIPs, indicating that ME1 and PAP, which share a similar folding pattern, can show different substrate specificity towards E. coli ribosomes. The results presented here are very significant, as few reports are available in the area of bacterial interaction with type I RIPs.