非经典的蛋白质分泌途径

蛋白质分泌是细胞间进行信息传递的重要方式之一。一般的分泌蛋白在其N端都具有信号肽序列,可以通过经典的内质网-高尔基体途径进行运输,并最终分泌到细胞外。近来发现细胞内存在另一类分泌蛋白,它们能够分泌到细胞外发挥功能,但却没有典型的信号肽序列,被称为非经典分泌蛋白。越来越多的证据表明,这类蛋白质的分泌有其独特的机制。本综述了这类蛋白质的各种分泌机制及可能的生理意义。     

枯草芽孢杆菌蛋白质分泌机制研究进展

综述了枯草芽孢杆菌不同蛋白质分泌机制,重点讨论了大多数细菌蛋白分泌的Sec途径,包括Sec途径的信号肽,信号肽酶,SecYEG通道,与分泌有关的各种细胞因子以及Sec途径的限制因素,此外还简要讨论了Tat途径,该途径能够转运折叠迅速或归密的蛋白质。     

过表达TatAdCd转位酶对枯草芽孢杆菌脂肪酶分泌的影响

【目的】研究过表达枯草芽孢杆菌Tat运输途径的Tat Ad Cd转位酶对促进脂肪酶分泌的影响。【方法】用cdd基因的串联启动子和前导区,替换tat AD-CD操纵子的启动子和前导区,并在染色体sac B基因位点整合表达;采用q RT-PCR方法表征tat AD-CD操纵子的表达水平;用脂肪酶表达质粒p HP13L转化Tat Ad Cd转位酶过表达菌株,构建产脂肪酶重组菌。通过测定脂肪酶活性,以及聚丙烯酰胺凝胶电泳,考察Tat Ad Cd转位酶过表达对脂肪酶分泌的影响。【结果】tat AD-CD操纵子被过表达,其胞内m RNA相对水平提高了185倍。Tat Ad Cd转位酶的过表达,使脂肪酶发酵单位提高了40%。【结论】使用cdd基因的串联启动子和前导区,能够有效地过表达目的基因;枯草芽孢杆菌脂肪酶可以同时经由Sec途径和Tat途径分泌;过表达Tat Ad Cd转位酶,能够显著提高脂肪酶的分泌量。     

基于多重计算设计策略提高枯草芽孢杆菌脂肪酶的热稳定性

提高酶的热稳定性是生物催化领域的热点和难点,计算机辅助的理性设计相比于传统的定向进化更加高效,在酶工程领域中的应用越来越广泛和深入。文中以枯草芽孢杆菌脂肪酶A为模式蛋白,首先,利用Rosetta-VIP计算设计对酶的结构空腔进行分析,选择了16个有利于结构空腔填充(ΔΔE<0)的单点突变,并以突变位点的溶剂可及表面积和进化保守性为二次筛选依据,测定了其热稳定性与酶活性。有6个单点突变体(F17A、V74I、L114P、I135V、M137A、I157L)的热稳定性得到了提高,其中Tm值最大提高3.18℃。结果表明,单点突变体满足ΔΔE越低、蛋白溶剂可及表面积减少且符合序列保守性,则得到保留原有酶活力的正向突变的可能性越大。此外,将热稳定性提高的6个单点突变进行迭代组合突变,两点组合突变体的Tm最大提高4.04℃,三点组合突变体的Tm最大提高5.13℃,四点组合突变体的Tm提高了7.30℃,六点组合突变体的Tm提高了7.43℃。因此,基于酶的分子结构的空腔分析、溶剂可及表面积及氨基酸序列保守性计算的多重虚拟筛选方法,可有效提高酶的热稳定性。     

SPNG+:基于stacking集成策略预测革兰氏阳性菌非经典分泌蛋白质

革兰氏阳性菌通过分泌毒力因子入侵宿主细胞引起化脓性炎症,进而导致疾病的产生,威胁人类健康.识别分泌蛋白有助于了解细菌分泌系统和致病机理,并为进一步筛选出毒力因子奠定基础.由于非经典分泌蛋白质缺乏经典信号肽序列,大规模实验鉴定此类蛋白质相对困难并且耗时耗力.目前,虽相继提出了一些计算预测方法,但它们对革兰氏阳性菌非经典分...     

真核细胞非经典蛋白分泌途径

在生物体中, 细胞间的信息传递是细胞生长、分化、发育、增殖、凋亡等生命活动的基本保证, 而蛋白分泌是细胞间信息传递的重要方式。大多数分泌蛋白都是通过内质网-高尔基体(ER-Golgi)途径分泌的。然而越来越多的研究表明, 存在着一类无信号肽的分泌蛋白, 这类蛋白不依赖ER-Golgi途径就能分泌到细胞外发挥功能, 被称为非经典分泌蛋白。非经典蛋白的分泌有其特有的机制, 它对ER-Golgi分泌途径是一种必要和有益的补充。非经典分泌与细胞增殖、免疫反应、肿瘤形成、传染病病理学等密切相关。文章旨在对非经典分泌蛋白的特点、分泌机制及生物学意义进行概述。     

利用短短小芽孢杆菌启动子和信号肽编码序列构建穿梭分泌表达载体

以短短小芽孢杆菌B15的总DNA为模板,利用PCR技术克隆到其细胞壁蛋白基因串联启动子和信号肽编码序列,测序分析后提交GenBank,登录号为AY956423。重新设计引物扩增该片段并在PCR产物两侧引入BamHⅠ和PstⅠ酶切位点,将PCR产物双酶切后克隆至穿梭载体pP43NMK的相应位点构建分泌表达载体pP15MK,插入片段置于该载体中mpd基因的上游,并使信号肽编码序列与去除了自身信号肽编码序列的mpd基因阅读框恰好融合。将pP15MK导入枯草杆菌构建表达菌株1A751(pP15MK),在短短小芽孢杆菌启动子和信号肽元件的带动下,mpd基因能够在表达菌株的对数生长期和稳定期持续性高效分泌表达,表达产物结合在细胞膜上;发酵液在48h酶活达到最高值7.79U/mL,是出发菌株邻单胞菌M6表达量的8.1倍。     

枯草芽孢杆菌(Bacillus subtilis)碱性脂肪酶基因在毕赤酵母中的表达

将来自枯草芽孢杆菌的碱性脂肪酶基因经密码子优化,全基因合成后克隆到pPICZαA载体,构建了pPICZαA-bsl分泌型重组质粒,该重组质粒经限制性内切酶PmeI线性化后使用LiCl法转化到毕赤酵母X-33,经过筛选获得分泌表达碱性脂肪酶的重组毕赤酵母X-33/pPICZαA-bsl。摇瓶发酵液上清酶活最高可达4.78 U/mL,初步研究了该脂肪酶的酶学性质,其最适作用温度为40-60℃,最适pH9.0,且具有高度耐碱的特性。该重组脂肪酶对旧新闻纸具备较明显的脱墨能力。     

非经典蛋白分泌研究进展

蛋白分泌是细胞间信息交流的重要途径。通常所指的蛋白分泌是经典分泌,即有信号肽的分泌蛋白会进入内质网,通过内质网-高尔基体运输释放。没有信号肽的胞质蛋白可以通过非经典的途径分泌,统称为非经典蛋白分泌。这些分泌蛋白在多个生物学过程中发挥作用,包括发育、代谢和炎症反应等,并且与人类重大疾病相关,如神经退行性疾病和癌症等。虽然对经典蛋白分泌途径已经有较深的了解,且主要内容已列入教科书,但是对非经典蛋白分泌的认识还处于初级阶段。本文就非经典蛋白分泌的研究进展作简要总结。     

乳酸菌蛋白质分泌表达研究进展

食品级乳酸菌不仅是食品或消化道中传递异源蛋白质的合适的候选菌,在工业发酵中还可用于生产蛋白质。在过去20多年中,人们设计了许多乳酸菌蛋白质表达和标记系统,这些系统已用在乳酸菌工程菌的细胞内或细胞外生产各种细菌、病毒和真核生物来源的蛋白质。在目的蛋白生产和发酵中,分泌表达由于可持续培养和简化纯化步骤并使目的蛋白与其靶位相互作用而优于细胞质表达。目前只有少数研究报道了目的蛋白在乳酸菌细胞内或分泌表达产量的比较,研究表明分泌表达比细胞质表达更优越。     

坚强芽胞杆菌主要分泌蛋白的鉴定及其分泌性序列

[目的]坚强芽胞杆菌是一种在自然界普遍存在的益生菌,在对虾养殖中应用较为广泛.为了研究其分泌性蛋白从而为分泌性载体的构建提供理论依据,本文对坚强芽胞杆菌的主要分泌蛋白进行质谱鉴定及分泌性序列的分析.[方法]从本实验室分离保存的1株来自对虾肠道的坚强芽胞杆菌(Bacillus firmus)培养液中获取了分泌性蛋白,进行SDS-PAGE,并对表达量较高的3条蛋白区带进行MALDI-TOF/TOF质谱鉴定及克隆测序,进行生物信息学分析.[结果]鉴定出的蛋白分别是坚强芽胞杆菌几丁质酶(chitinase)、坚强芽胞杆菌肠毒素A(enterotoxin A)和坚强芽胞杆菌BCG9842蛋白(hypothetical protein BCG9842).经在线软件SignaIP 3.0分析,确定chitinase、enterotoxin和BCG9842均存在不同的分泌性信号肽序列bf-43、bf-37和bf-16,通过在线软件PSORT分析表明,bf-43定位于细胞的外膜上,bf-37和bf-16定位于细胞的胞外.[结论]本研究鉴定出了坚强芽胞杆菌的3个主要分泌性蛋白,分析筛选出了3条分泌性序列,为分泌性载体的构建提供理论依据.     

马铃薯晚疫病菌全基因组分泌蛋白的初步分析

利用马铃薯晚疫病菌全基因组测序结果,结合计算机技术和生物信息学的方法,对马铃薯晚疫病菌的蛋白进行分析,为明确该病原菌与寄主互作的分子机制奠定基础。文章应用信号肽预测软件SignalP v3.0和PSORT,跨膜螺旋结构预测软件TMHMM-2.0和THUMBUP,GPI锚定位点预测软件big-PI Predictor,亚细胞器中蛋白定位分布预测软件TargetP v1.01,对已经公布的马铃薯晚疫病菌全基因组22 658个蛋白质氨基酸序列进行分析。结果发现,晚疫病菌全基因组编码蛋白中有671个为潜在的分泌型蛋白,占编码蛋白总数的3.0%。其中有45个分泌蛋白有功能方面的描述,其功能涉及细胞代谢、信号转导等方面;此外,还有一些与激发子类似的分泌蛋白,它们可能与晚疫病菌的毒性有关。     

Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.     

Combined analysis of secreted proteins and glycosylation identifies prognostic features in cholangiocarcinoma

Secreted proteins are overexpressed in cholangiocarcinoma (CCA) and actively involved in promoting metastatic spread. Many of these proteins possess one or more sites of glycosylation and their various glycoforms have potential utility as prognostic or diagnostic biomarkers. To evaluate the effects of secretome glycosylation on patient outcome, we elucidated the glycosylation patterns of proteins secreted by parental and metastatic CCA cells using liquid chromatography-mass spectrometry. Our analysis showed that the secretome of CCA cells was dominated by fucosylated and fucosialylated glycoforms. Based on the glycan and protein profiles, we evaluated the combined prognostic significance of glycosyltransferases and secretory proteins. Significantly, genes encoding fucosyltransferases and sialyltransferases showed favorable prognostic effects when combined with secretory protein-coding gene expression, particularly thrombospondin-1. Combining these measures may provide improved risk assessment for CCA and be used to indicate stages of disease progression.     

优雅蝈螽雄性附腺结构与分泌蛋白特性

通过组织切片和SDS-PAGE方法,研究优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl雄性附腺的结构及分泌蛋白的特性。结果表明,优雅蝈螽雄性附腺由3类腺管组成:乳白长腺管、透明腺管和乳白短腺管,腺管的管壁组织结构相似,从内到外依次为单层上皮细胞、基膜、肌肉层,不同腺管管腔分泌物H-E染色后呈现不同颜色。SDS-PAGE分析各种腺管的分泌蛋白具有特异性。     

A method for the identification of proteins secreted by lactic acid bacteria grown in complex media

Lactic acid bacteria (LAB) are known for their special nutritional requirements, being usually cultured in complex media to achieve optimal growth. In this paper, a protocol based on trichloroacetic acid precipitation of peptides and proteins is presented. The method has been tested on four probiotic LAB strains grown in De Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the culture of such bacteria. This protocol allowed the detection of 19 proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being successfully identified by tandem MS. Thereafter, the 10 were found to be secreted or surface associated by bioinformatic means. In conclusion, this work supplies a method for the identification of proteins secreted by LAB, allowing discrimination between the proteins present in the MRS and those produced by probiotic LAB.     

Effect of environmental conditions on proteins secreted by enterohemorrhagic Escherichia coli O26:H11

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal diseases in humans, and these bacteria have recently emerged as a leading cause of renal failure and encephalitis in children and the aged. In this study, we examined the environment-dependent production of proteins secreted from a strain of STEC O26:H11 by trichloroacetic acid precipitation, SDS-PAGE, Western blotting and N-terminal amino acid sequence analysis. Growth of bacteria in essential minimum medium (M9) led to the detection of secreted proteins of 104, 80,40, 37 and 25 kDa (P104, P80, P40, P37 and P25, respectively). When grown in serum-free MEM, only P104, P40, P37 and P25 were observed in supernatant fluids. Growth of the bacteria in Luria-Bertani broth (LB) enhanced the expression of P104, but the productions of the other proteins were remarkably reduced. CO2 increased the secretion of P80 and P37, but reduced the production of P104. N-terminal amino acid sequencing revealed that P104 was EspP of STEC, which was homologous to EspC of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a subclass of the IgA protease family. P80, which was identified as EspE of STEC, was homologous to Tir of EPEC. P40, P37 and P25 were found to be highly homologous to the similarly sized EspD, EspB and EspA proteins, previously detected in culture supernatants of EPEC. Those proteins are thought to be STEC virulence factors. Sera were obtained from two patients, one with colitis and another with hemolytic uremic syndrome (HUS), caused by STEC O157:H7, to study immune response to secreted proteins. Our results suggested that Tir caused immune response following STEC disease.     

iNSP-GCAAP: Identifying nonclassical secreted proteins using global composition of amino acid properties

Nonclassical secreted proteins (NSPs) refer to a group of proteins released into the extracellular environment under the facilitation of different biological transporting pathways apart from the Sec/Tat system. As experimental determination of NSPs is often costly and requires skilled handling techniques, computational approaches are necessary. In this study, we introduce iNSP-GCAAP, a computational prediction framework, to identify NSPs. We propose using global composition of a customized set of amino acid properties to encode sequence data and use the random forest (RF) algorithm for classification. We used the training dataset introduced by Zhang et al. (Bioinformatics, 36(3), 704–712, 2020) to develop our model and test it with the independent test set in the same study. The area under the receiver operating characteristic curve on that test set was 0.9256, which outperformed other state-of-the-art methods using the same datasets. Our framework is also deployed as a user-friendly web-based application to support the research community to predict NSPs.     

Construction of second generation protease-deficient hosts of Bacillus subtilis for secretion of foreign proteins

Although one of the major factors limiting the application of Bacillus subtilis as an expression host has been its production of at least eight extracellular proteases, researchers have also noticed that some proteases benefited the secretion of foreign proteins at times. Therefore, to maximize the yield of a foreign protein, the proteases should be selectively inactivated. This raises a new question that how to identify the favorable and unfavorable proteases for a target protein. Here, an evaluation system containing nine mutant strains of B. subtilis 168 was developed to address this question. The mutant strain PD8 has all the eight proteases inactivated whereas each of the other eight mutant strains expresses only one kind of these eight proteases. The target protein is secreted in these nine mutant strains; if the production of target protein in a mutant strain is higher than that in strain PD8, the corresponding protease is regarded as favorable. Accordingly, the optimal protease-deficient host is constructed through inactivating the unfavorable proteases. The effectiveness of this system was confirmed by expressing three foreign proteins. This study provides a strategy for improving the secretion of a foreign protein in B. subtilis through tailoring a personalized protease-deficient host.