大肠杆菌Tat蛋白质转运体系

Tat是大肠杆菌中能够将折叠蛋白质跨膜转运的体系,其信号肽中含有一个高度保守的双精氨酸模体。Tat体系包括TatA、TatB、TatC和TatE4种蛋白质,它们的复合物在大肠杆菌质膜上形成转运通道。大肠杆菌Tat体系转运的底物蛋白质多为呼吸电子传递链组分,与大肠杆菌的许多生命活动有关。     

大肠杆菌Tat转运酶的研究进展

TatA、TatB和TatC是大肠杆菌Tat转运酶的组成成分.研究表明各Tat蛋白具有不同的功能区域, TatA和TatB蛋白功能重要的位点位于N末端的穿膜片断、其后的双极性α-螺旋和铰链区.TatC的序列保守性低,N末端穿膜片断和位于胞质内的第一环区对转运是必需的.Tat转运酶各成分相互结合成复合物形式并相互依赖.TatA在细胞中高表达并自身聚合形成数量不等的同聚物,具有稳定TatBC复合物的作用,TatB有稳定TatC的功能,TatB和TatC两者结合形成二聚体.实验表明,TatA复合物形成转运通道,TatBC复合物通过TatC蛋白识别底物的信号肽并与底物结合, 再在TatB介导下与TatA复合物结合形成具有活性的转运酶.     

细菌蛋白质Tat转运系统的研究进展

蛋白质Tat转运系统不同于细菌中普遍存在的Sec转运系统,而与植物叶绿体中蛋白质转运的ΔpH依赖系统相似.通过Tat系统转运的蛋白质底物含有特征性的双精氨酸保守序列核心S/T-R-R-x-F-L-K的信号肽,其h区的疏水性低,c区有由高赖氨酸、高精氨酸构成的避开Sec系统信号,信号肽和成熟蛋白质的组成对蛋白质的转运都有影响.TatA、TatB、TatC和TatE四种蛋白质参与了大肠杆菌的Tat转运系统.被转运的底物蛋白质绝大多数为与细菌厌氧呼吸有关的含氧化还原辅因子的酶,并以折叠形式转运.     

一株Tat蛋白质转运系统受阻菌株特性的研究

大肠杆菌野生型菌株MC4100A经低能氮离子注入处理后,根据细胞排列方式定向筛选到了一株突变菌株ZSY。ZSY与MCAl00A相比有很大的差异,细胞呈链状排列,丧失了细胞分裂后分离的能力;2％SDS对ZSY有一定的杀伤性;在厌氧状态下,失去了以甘油为碳源和TMAO为电子受体的生长能力。结果表明：突变株ZSY具有的一系列性状同编码双精氨酸转运系统受阻的菌株相同,因而ZSY的突变很可能发生在与细菌蛋白质Tat转运系统相关的基因上。特别有意义的是突变菌株在37℃下生长缓慢,难以形成菌落,而在4℃放置3天,可形成直径为0．5mm的菌落。     

带Tat标记质粒载体的构建及其转导融合蛋白进入细胞的研究

采用点突变技术构建了带 6×His、Tat和Flag多个标记的pET HTF的质粒载体 ,利用基因重组技术构建pET HTF EGFP融合蛋白载体 .酶切和DNA测序证明 ,所构建的pET HTF和pET HTF EGFP载体正确 .BL2 1(DE3)表达融合蛋白 ,用Ni2 + 分离柱纯化His Tat Flag EGFP蛋白 ,并加入培养的NIH3T3细胞 .荧光显微镜观察显示 ,His Tat Flag EGFP融合蛋白进入细胞 .带His、Tat和Flag标记的质粒载体pET 14b HTF表达的融合蛋白能够进入细胞 ,该载体为进行蛋白质功能研究和基因治疗研究提供了一个重要工具     

绿色荧光蛋白在蛋白质研究中的应用

绿色荧光蛋白（green fluorescent protein,GFP）自发现以来,由于具有自发荧光等特性,在分子生物学和细胞生物学领域得到广泛应用。GFP作为一种报道分子,在研究蛋白质相互作用和构象变化、检测蛋白质表达、蛋白质和细胞荧光示踪中,起到了重要的作用。该文通过对绿色荧光蛋白特性的分析．介绍其作为荧光标记在蛋白质研究中的应用,并展望进一步的研究前景。     

蛋白质向叶绿体的转运

对近年来叶绿体蛋白质前导肽序列、叶绿体被膜中的蛋白质转运器、监护蛋白在蛋白转运过程中的作用、蛋白质导入叶绿体的途径、前体蛋白的加工的研究进展进行了介绍和评述     

基于蛋白质跨外膜自转运系统的细菌细胞表面蛋白展示技术研究进展

革兰氏阴性菌Ⅴ型分泌系统是细菌病原蛋白分泌的主要途径之一,可分为Ⅴa-Ⅴe5个亚型,其中Ⅴa型(即经典的单体自转运蛋白)是细菌毒力和黏附因子向细胞外分泌的重要工具,其在内膜Sec易位子和外膜BAM蛋白复合体的协助下,通过2个连续的跨膜步骤介导蛋白质穿过阴性菌的内外膜.据信Va型是目前已知蛋白质跨膜转运时最简单的分泌途径...     

植物细胞质介导GFP-NLS蛋白入核转运的HeLa细胞系统的建立

细胞核作为细胞中重要的遗传物质存储、复制和转录的结构,牵涉着大量信息和物质的传输活动,尤其是蛋白质的入核转运一直以来都是研究的热点问题之一。本文利用病毒SV40抗原蛋白中的核定位信号（nuclear localization signal,NLS）标记GFP蛋白,通过拟南芥细胞质的介导,利用HeLa细胞核建立起了研究蛋白质入核转运的半细胞体系。结果显示,植物细胞质结合NLS片段能改变GFP在HeLa细胞核内外的分布,实现对目标蛋白入核过程的介导,使GFP-NLS最后定位于细胞核内。这也意味着通过HeLa细胞建立起的半细胞体系能为蛋白入核转运研究提供一个有效的研究体系。     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

Unusual signal peptide directs penicillin amidase from Escherichia coli to the Tat translocation machinery

The recently described Tat protein translocation system in Escherichia coli recognizes its protein substrates by the consensus twin arginine (SRRXFLK) motif in the signal peptide. The signal sequence of E. coli pre-pro-penicillin amidase bears two arginine residues separated by one aspargine and does not resemble the Tat-targeting motif but can nevertheless target the precursor to the Tat pathway. Mutational studies have shown that the hydrophobic core region acts in synergism with the positive charged N-terminal part of the signal peptide as a Tat recognition signal and contributes to the efficient Tat targeting of the pre-pro-penicillin amidase.     

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.     

The Tat system proofreads FeS protein substrates and directly initiates the disposal of rejected molecules

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane, including FeS proteins that receive their cofactors in the cytoplasm. We have studied two Escherichia coli Tat substrates, NrfC and NapG, to examine how, or whether, the system exports only correctly folded and assembled FeS proteins. With NrfC, substitutions in even one of four predicted FeS centres completely block export, indicating an effective proofreading activity. The FeS mutants are rapidly degraded but only if they interact with the Tat translocon; they are stable in a tat deletion strain and equally stable in wild-type cells if the signal peptide twin-arginine motif is removed to block targeting. Basically similar results are obtained with NapG. The Tat apparatus thus proofreads these substrates and directly initiates the turnover of rejected molecules. Turnover of mutated FeS substrates is completely dependent on the TatA/E subunits that are believed to be involved in the late stages of translocation, and we propose that partial translocation triggers substrate turnover within an integrated quality control system for FeS proteins.     

绿色荧光蛋白与 HCV 核心蛋白的融合及在大肠杆菌中的高效表达

利用基因工程重组技术获得了绿色荧光蛋白（ｇｆｐ）基因与ＨＣＶ核心蛋白基因的嵌合体,并在大肠杆菌中高效表达了４８ｋＤａ的融合蛋白,经Ｄｏｔ－ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ ｂｌｏｔ免疫活性分析证实,融合蛋白仍具有ｃｏｒｅ抗原的三个免疫活性部位,同时用荧光显微镜观察并用荧光光度计测定了大肠直菌表达的融合蛋白的荧光光谱,结果证实,我们在大肠杆菌中表达的ＧＦＰ－ｃｏｒｅ融合蛋白既能发射易于检测的绿色荧光,又具     

Protein secretion in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for the industrial production of amino acids, such as glutamate and lysine, for decades. Due to several characteristics – its ability to secrete properly folded and functional target proteins into culture broth, its low levels of endogenous extracellular proteins and its lack of detectable extracellular hydrolytic enzyme activity – C. glutamicum is also a very favorable host cell for the secretory production of heterologous proteins, important enzymes, and pharmaceutical proteins. The target proteins are secreted into the culture medium, which has attractive advantages over the manufacturing process for inclusion of body expression – the simplified downstream purification process. The secretory process of proteins is complicated and energy consuming. There are two major secretory pathways in C. glutamicum, the Sec pathway and the Tat pathway, both have specific signal peptides that mediate the secretion of the target proteins. In the present review, we critically discuss recent progress in the secretory production of heterologous proteins and examine in depth the mechanisms of the protein translocation process in C. glutamicum. Some successful case studies of actual applications of this secretory expression host are also evaluated. Finally, the existing issues and solutions in using C. glutamicum as a host of secretory proteins are specifically addressed.     

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.     

Mutations in subunits of the Escherichia coli twin-arginine translocase block function via differing effects on translocation activity or tat complex structure

We have used a combination of blue-native (BN) gel electrophoresis and protein purification to analyze the effects of TatA or TatC mutations on the structures of the primary TatABC and multimeric TatA complexes in Escherichia coli. Expression of wild-type TatABC leads to the production of a single major TatABC complex of 370 kDa and a heterogeneous set of TatA complexes of <100 kDa to approximately 500 kDa. Two TatC mutations that block translocation have different effects on complex structures. P48A causes massive defects in TatABC assembly, including a marked separation of the TatBC subunits and the production of TatB and TatC aggregates. In contrast, TatABC complexes from the inactive TatC F94A mutant are structurally intact, suggesting that this mutation affects translocation activity rather than assembly. Neither TatC mutation affects the separate TatA complexes, showing that assembly of the TatA complexes is independent of TatABC assembly or activity. In contrast, three TatA mutations affect both the TatA and TatABC complexes. F39A assembles into smaller, incorrectly organized TatA complexes and the TatABC complexes contain an incorrect TatB:TatC ratio and unusually large amounts of TatA. A triple mutant in the amphipathic region forms slightly larger TatA complexes that are likewise disorganized, and a mutant containing three glycine substitutions in the transmembrane (TM) span assembles as grossly affected TatA complexes that are much larger than wild-type complexes. These mutants lead to a partial failure of TatB to assemble correctly. The data show that the amphipathic and TM regions play critical roles in TatA complex assembly. All of the TatA mutations lead to partial or substantial defects in TatABC complex formation, demonstrating that the properties of TatA can have a marked influence on the TatABC complex.