天然活性多肽的研究进展

综述了近年来天然来源的活性多肽的研究近况,主要侧重直链肽的研究近况,包括人,动物,植物来源多肽的提取分离方法,氨基酸组成以及生物活性等方面的研究进展。     

微生物异源合成植物异喹啉生物碱的新进展

植物异喹啉生物碱(plant isoquinoline alkaloids,PIAs)包括吗啡、可待因、加兰他敏及小糵碱等药用活性产物和其他天然活性产物。从植物中提取异喹啉生物碱,受制于低含量、种植季节及提取方法。人们开始研究利用微生物异源合成和改造天然异喹啉生物碱,从而获得低成本的药用活性物质。异喹啉生物碱合成途径长,反应复杂,为实现微生物异源合成带来了诸多挑战。随着合成途径和酶的解析和鉴定,合成生物学技术为在微生物中合成异喹啉生物碱提供了可能。综述了PIAs合成途径解析的最新进展,以及微生物异源合成PIAs的代谢工程策略,讨论了目前存在的问题和未来的发展趋势。     

HCV丝氨酸蛋白酶小分子底物构建及GST融合表达

丝氨酸蛋白酶是丙型肝炎病毒重要的功能蛋白和药物作用靶点,其通过分子内（cis）和分子间（trans）方式催化水解前体蛋白,释放病毒功能蛋白。目的：为深入研究病毒蛋白酶活性和抑制剂鉴定需要,实验研究参照丙型肝炎病毒1a亚型菌株蛋白酶天然底物的氨基酸序列特点,设计了一段包含两个天然底物酶切位点的小分子多肽2S,并进行了原核表达。方法：利用PCR方法,合成2S小分子多肽基因,目的基因两端引入BamH I和EcoR I两个限制性酶切位点,双酶切后将基因与表达载体pGEX-4T-2重组,转化大肠杆菌DH5α,经化学诱导进行GST融合蛋白表达,通过亲和层析柱纯化目的蛋白。纯化的GST 2S融合蛋白在体外反应系统进行酶切鉴定,SDS-PAGE和ELISA鉴定酶切结果。结果：PCR合成的小分子底物多肽2S基因,经与表达载体重组后测序,证实基因序列正确。采用0.5mmol/L浓度的IPTG诱导工程菌过夜,获得表达的目的蛋白,经分离纯化得到融合蛋白GST-2S。GST-2S在体外磷酸盐缓冲系统中与丝氨酸蛋白酶反应,15%SDS-PAGE鉴定酶切产物,证实融合蛋白底物条带明显消失,ELISA结果同样说明融合蛋白的底物活性。结论：含有两个天然底物酶切位点的小分子多肽可以替代病毒天然底物,实验结果为丙型肝炎病毒丝氨酸蛋白酶活性研究和酶抑制剂研究奠定了方法学基础。     

乙内酰脲酶及其在氨基酸手性合成中的应用

乙内酰脲水解酶、氨甲酰化酶和乙内酰脲消旋酶构成的酶系能够以5-取代乙内酰脲类化合物为原料合成天然和非天然D-或L-氨基酸,用于各种手性氧基酸的生产。近来的研究重点在分离新酶或提高原酶的活性,包括定向突变、三维结构解析与结构功能关系研究、酶固定化、蛋白融合和构建完整细胞生物催化剂等。     

蛋白质组学分析技术在微生物研究中的应用

随着后基因组时代的到来,蛋白质组学分析为研究微生物的生命活动和细胞功能提供了一个广阔的视角。综述了大规模分析微生物蛋白质组的策略和方法,包括自上而下的蛋白质组学分析、自下而上的蛋白质组学分析、蛋白质组定量分析技术、蛋白质修饰研究方法和蛋白质芯片技术。最后,对沙门氏菌蛋白质组学的研究进展进行了简要介绍。     

代谢工程改造酿酒酵母合成植物萜类D-柠檬烯的策略

植物萜类化合物是以异戊二烯为结构单位的一大类植物天然的次生代谢产物。D-柠檬烯属于单萜类化合物,由于它具有抑菌、增香、抗癌、止咳、平喘等多种功能,已被广泛应用于食品、香料、医疗等行业。目前D-柠檬烯的工业生产主要是从植物的果皮或者果肉中提取的,但提取方法存在着分离纯化复杂、产率低、能耗大等缺点。而本世纪初合成生物学技术的兴起,为微生物异源合成天然活性化合物带来了全新的理念与工具,打破了物种间的界限,使微生物异源合成D-柠檬烯成为现实。构建定向、高效的异源合成D-柠檬烯的微生物细胞工厂,实现微生物发酵法替换传统的植物提取法,具有重要的经济与社会效益。本文主要回顾了近几年利用代谢工程改造酿酒酵母异源合成萜类化合物取得的成就,阐述了以酿酒酵母作为底盘微生物,利用代谢工程和合成生物学的手段构建高产D-柠檬烯的合成策略。     

微生物合成根皮素及其糖苷研究进展 *

根皮素及其糖苷是二氢查耳酮类天然产物,具有抗氧化、抗炎和抑菌等多种生理活性,可应用于食品、药品、化妆品等行业。目前,根皮素及其糖苷主要从植物中提取,但含量低、组分复杂制约了高效、低成本的分离制备。随着合成生物学的发展,工程微生物生产根皮素及其糖苷将是一种有潜力的方法。综述了微生物合成根皮素及其糖苷的关键基因鉴定、代谢途径重构和优化策略,并针对酶的特异性差、副产物多等问题提出了技术对策。     

酵母 tRNA~(ala)密码子GpCpU的酶促合成

tRNA主要有两种生物学功能:一是接受(相应的氨基酸。二是将此氨基酸转移到多肽链中。在后一功能中,tRNA通过其反密码子同mRNA上相应的密码子形成互补碱基间的氢键配对,从而使氨基酸转移到由mRNA碱基顺序决定的多肽序列中。本工作合成酵母tRNA~(ala)密码子GpCpU,将用于测验该tRNA的转移活性,即检查该tRNA能否通过其反密码子3＇CpGpI5＇同已结合在核糖体上的密码子5＇GpCpU3＇形成氢键配对而实现转移丙氨酸的功能。迄今报道的关于制备寡核苷酸的方法,主要有三种:化学合成、酶解天然核酸和酶促合成。本工作用最后一种方法,用RN_(ase)N_1和     

蜂毒肽片段的合成及其与钙调蛋白的相互作用

采用固相法设计合成了４个蜂毒肽片段：Ｍｅｌ１２、Ｍｅ１１３、Ｍｅｌ１４、Ｍｅｌ１５。应用电泳技术,抑制钙依赖性的磷酸二酯酶酶活方法和荧光技术研究了这些多肽与钙调蛋白的相互作用。结果表明这些多肽与钙调蛋白均形成１：１复合物,抑制钙依赖性的磷酸二酯酶的活性,其中Ｍｅｌ１４和Ｍｅｌ１５对钙调蛋白的结合活性与完整的蜂毒肽比较接近。     

多肽固相合成的研究进展

多肽固相化学合成法是蛋白质研究领域非常重要的研究方法之一,主要可以分为Boc方法和Fmoc方法,在生物药物、蛋白质工程、免疫学等研究中得到了广泛的应用。该文论述了多肽固相合成法的原理,比较了两种典型合成方法的优缺点,介绍了适用于该方法的多肽种类,提出了多肽合成过程中存在的问题与解决策略,最后展望了多肽合成法的应用前景,以供相关领域的研究人员参考。     

Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.     

Practical 4'-phosphopantetheine active site discovery from proteomic samples

Polyketide and nonribosomal peptides constitute important classes of small molecule natural products. Due to the proven biological activities of these compounds, novel methods for discovery and study of the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymes responsible for their production remains an area of intense interest, and proteomic approaches represent a relatively unexplored avenue. While these enzymes may be distinguished from the proteomic milieu by their use of the 4'-phosphopantetheine (PPant) post-translational modification, proteomic detection of PPant peptides is hindered by their low abundance and labile nature which leaves them unassigned using traditional database searching. Here we address key experimental and computational challenges to facilitate practical discovery of this important post-translational modification during shotgun proteomics analysis using low-resolution ion-trap mass spectrometers. Activity-based enrichment maximizes MS input of PKS/NRPS peptides, while targeted fragmentation detects putative PPant active sites. An improved data analysis pipeline allows experimental identification and validation of these PPant peptides directly from MS2 data. Finally, a machine learning approach is developed to directly detect PPant peptides from only MS2 fragmentation data. By providing new methods for analysis of an often cryptic post-translational modification, these methods represent a first step toward the study of natural product biosynthesis in proteomic settings.     

Creation of Functional Micro/Nano Systems through Top-down and Bottom-up Approaches

Mimicking nature's approach in creating devices with similar functional complexity is one of the ultimate goals of scientists and engineers. The remarkable elegance of these naturally evolved structures originates from bottom-up self-assembly processes. The seamless integration of top-down fabrication and bottom-up synthesis is the challenge for achieving intricate artificial systems. In this paper, technologies necessary for guided bottom-up assembly such as molecular manipulation, molecular binding, and the self assembling of molecules will be reviewed. In addition, the current progress of synthesizing mechanical devices through top-down and bottom-up approaches will be discussed.     

Identification and validation of protein targets of bioactive small molecules

Identification and validation of protein targets of bioactive small molecules is an important problem in chemical biology and drug discovery. Currently, no single method is satisfactory for this task. Here, we provide an overview of common methods for target identification and validation that historically were most successful. We have classified for the first time the existing methods into two distinct and complementary types, the 'top-down' and 'bottom-up' approaches. In a typical top-down approach, the cellular phenotype is used as a starting point and the molecular target is approached through systematic narrowing down of possibilities by taking advantage of the detailed existing knowledge of cellular pathways and processes. In contrast, the bottom-up approach entails the direct detection and identification of the molecular targets using affinity-based or genetic methods. A special emphasis is placed on target validation, including correlation analysis and genetic methods, as this area is often ignored despite its importance.     

Microbial production of small peptide: pathway engineering and synthetic biology

Small peptides are a group of natural products with low molecular weights and complex structures. The diverse structures of small peptides endow them with broad bioactivities and suggest their potential therapeutic use in the medical field. The remaining challenge is methods to address the main limitations, namely (i) the low amount of available small peptides from natural sources, and (ii) complex processes required for traditional chemical synthesis. Therefore, harnessing microbial cells as workhorse appears to be a promising approach to synthesize these bioactive peptides. As an emerging engineering technology, synthetic biology aims to create standard, well-characterized and controllable synthetic systems for the biosynthesis of natural products. In this review, we describe the recent developments in the microbial production of small peptides. More importantly, synthetic biology approaches are considered for the production of small peptides, with an emphasis on chassis cells, the evolution of biosynthetic pathways, strain improvements and fermentation.     

Microscale methods to assemble mammalian cells into tissue-like structures

Different cell types make up tissues and organs hierarchically and communicate within a complex, three-dimensional (3D) environment. The in vitro recapitulation of tissue-like structures is meaningful, not only for fundamental cell biology research, but also for tissue engineering (TE). Currently, TE research adopts either the top-down or bottom-up approach. The top-down approach involves defining the macroscopic tissue features using biomaterial scaffolds and seeding cells into these scaffolds. Conversely, the bottom-up approach aims at crafting small tissue building blocks with precision-engineered structural and functional microscale features, using physical and/or chemical approaches. The bottom-up strategy takes advantage of the repeating structural and functional units that facilitate cell-cell interactions and cultures multiple cells together as a functional unit of tissue. In this review, we focus on currently available microscale methods that can control mammalian cells to assemble into 3D tissue-like structures.     

Interaction profiling methods to map protein and pathway targets of bioactive ligands

Recent advances in -omic profiling technologies have ushered in an era where we no longer want to merely measure the presence or absence of a biomolecule of interest, but instead hope to understand its function and interactions within larger signaling networks. Here, we review several emerging proteomic technologies capable of detecting protein interaction networks in live cells and their integration to draft holistic maps of proteins that respond to diverse stimuli, including bioactive small molecules. Moreover, we provide a conceptual framework to combine so-called ‘top-down’ and ‘bottom-up’ interaction profiling methods and ensuing proteomic profiles to directly identify binding targets of small molecule ligands, as well as for unbiased discovery of proteins and pathways that may be directly bound or influenced by those first responders. The integrated, interaction-based profiling methods discussed here have the potential to provide a unique and dynamic view into cellular signaling networks for both basic and translational biological studies.     

Relative roles of top-down and bottom-up forces in terrestrial tritrophic plant–insect herbivore–natural enemy systems

Whether resources (bottom-up forces), natural enemies (top-down forces), or both, determine the abundance of insect herbivore populations in plant–insect herbivore–natural enemy systems remains a major issue in population ecology. Unlike recent surveys of the tritrophic literature we do not seek to quantify whether top-down or bottom-up forces predominate in any given set of experimental systems. Acknowledging the dearth of empirical synthesis we employ two contrasting literature surveys to determine whether the plant–insect herbivore–natural enemy literature is currently adequate to form a conceptual synthesis of the relative roles of top-down and bottom-up forces. The emergence of a synthesis of the relative roles of top-down and bottom-up forces in plant–insect herbivore–natural enemy systems appears to have been largely prevented by (1) the absence of appropriate empirical data; (2) failure to appreciate the merits of existing data; (3) a continued desire to emphasise either top-down or bottom-up forces to the exclusion of the other; and (4) confusion regarding which processes regulate and which influence the abundance of insect herbivores.