Yeast "N"-hybrid systems for protein-protein and drug-protein interaction discovery

The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and characterize protein-protein interactions, but recent modifications and improvements have opened up new routes towards screening for small molecule-protein interactions. Such yeast "n"-hybrid systems hold great promise for the development of drugs which interfere with protein-protein interactions and for the discovery of drug-target interactions. In this review, we discuss several yeast two-hybrid based approaches with applications in drug discovery and describe a protocol for yeast three-hybrid screening of small molecules to identify their direct targets.     

A combined yeast/bacteria two-hybrid system: development and evaluation

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.     

用于蛋白质间相互作用研究的新型双杂交系统

近年来,一些不依赖于转录因子活性的新型双杂交系统相继建立,如分离的泛素系统、蛋白质片段互补分析、阻遏物重构分析和SOS恢复系统等。同利用转录因子活性的酵母双杂交系统相似,这些方法也利用了一些活性蛋白的结构与功能特点来研究蛋白质间相互作用,这些活性蛋白不是转录因子,但也可在结构上进行分离可通过重构使其生物活性得以恢复。由于这些新型双杂交系统的各自特点,使得它们成为酵母双杂交系统的有益补充和研究蛋白质间相互作用的有力工具。     

The application of yeast hybrid systems in protein interaction analysis

Yeast hybrid systems have been widely used due to their convenience and low cost. Based on these systems, many methods have been developed to analyze protein–protein, protein–DNA and protein–RNA interactions. In this paper, we are reviewing these different yeast hybrid systems. According to the number of hybrid proteins, yeast hybrid systems can be divided into three categories, yeast one-hybrid, yeast two-hybrid and yeast three-hybrid systems. Alternatively, yeast hybrid systems can be categorized according to the subcellular localization of the protein interaction process in the cell into nuclear protein–protein interactions, cytosol protein–protein interactions and membrane protein–protein interactions. Throughout the review, we focus on the progress and limitations of each yeast hybrid system over the recent years.     

Protein recruitment systems for the analysis of protein +/- protein interactions

The yeast Saccharomyces cerevisiae serves as an excellent genetic tool for the analysis of protein +/- protein interactions. The most common system, used to date, is the two-hybrid system. Although proven very powerful, the two-hybrid system exhibits several inherent problems and limitations. Recently, two alternative systems have been described that take advantage of the fact that localization of signal transduction effectors to the inner leaflet of the plasma membrane is absolutely necessary for yeast viability. These effectors can either be the Ras guanyl nucleotide exchange factor or Ras itself. The yeast strain used in both systems is a temperature-sensitive mutant in the yeast Ras guanyl nucleotide exchange factor, CDC25. Membrane localization of these effectors is achieved via protein +/- protein interaction. Each system can be used to test interaction between known protein pairs, as well as for isolation of novel protein interactions. Described here are the scientific and technical steps to be considered for both protein recruitment systems.     

Bacterial expression systems for recombinant protein production: E. coli and beyond

Escherichia coli expression system continues to dominate the bacterial expression systems and remain to be the preferred system for laboratory investigations and initial development in commercial activities or as a useful benchmark for comparison among various expression platforms. Some new developments in overcoming its shortcomings are reviewed in this paper, including antibiotics-free selection plasmids, extracellular production, and posttranslational modifications. The ability for E. coli to make mg glycosylated proteins promises even broader applications of the E. coli system in the future. Significant progresses have also been made over the past few years in alternative bacterial expression systems. Notably, the Lactoccocus lactis system has proven to be a viable choice for membrane proteins. Additionally, several Pseudomonas systems were developed and achieved product titers comparable to E. coli systems. Other bacterial systems such as Streptomyces, coryneform bacteria, and halophilic bacteria offer advantages in some niche areas, providing more choices of bacterial expression systems for recalcitrant proteins.     

Organic transformations catalyzed by engineered yeast cells and related systems

Asymmetric ketone reductions remain the most popular application of baker's yeast (Saccharomyces cerevisiae) in organic synthesis and data from the genome sequencing project is beginning to have an impact on improving the stereoselectivities of these reactions, augmenting traditional approaches based on selective inhibition. In addition, the catalytic repertoire of yeast has been expanded to include chiral ketone oxidations by overexpression of a bacterial Baeyer-Villiger monooxygenase.     

Split-protein systems: beyond binary protein-protein interactions

It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches.     

A yeast assay for high throughput screening of natural anti-viral agents

Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions. Many variations of these basic systems have originated over the years. Besides these vast and varied applications of the yeast expression system, S. cerevisiae has also been used extensively in fundamental research as a model simple eukaryote. We have used the S. cerevisiae system to design a high throughput screen for anti-viral agents from natural sources. The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting. A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate. Using such a process, we have initiated a high throughput screening process for natural anti-viral agents.     

Genomic analysis of secretion systems

Secretion of proteins into the extracellular environment is important to almost all bacteria, and in particular mediates interactions between pathogenic or symbiotic bacteria with their eukaryotic hosts. The accumulation of bacterial genome sequence data in the past few years has provided great insights into the distribution and function of these secretion systems. Three systems are responsible for secretion of proteins across the bacterial cytoplasmic membrane: Sec, SRP and Tat. Many novel examples of systems for transport across the Gram-negative bacterial cell envelope have been discovered through genome sequencing and surveys, including many novel type III secretion systems and autotransporters. Similarly, genomic data mining has revealed many new potential secretion substrates and identified unsuspected domains in secretion-associated proteins. Interestingly, genomic analyses have also hinted at the existence of a dedicated protein secretion system in Gram-positive bacteria, targeting members of the WXG100/ESAT-6 family of proteins, and have revealed an unexpectedly wide distribution of sortase-driven protein-targeting systems.     

Production of serpins using yeast expression systems

Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered.     

酵母双杂交系统的发展及其衍生系统的比较

酵母双杂交及其衍生系统是鉴定及分析蛋白质-蛋白质、蛋白质-DNA、蛋白质-RNA相互作用的最常用、最有效的工具之一。本文系统介绍了该技术的背景、发展过程,以及由Fields和Song初次描述的由酵母双杂交系统衍生而来的几种主要的双杂交系统的特点,并简要比较了各系统的优缺点。     

Rapid detection of protein tyrosine kinase activity in recombinant yeast expressing a universal substrate

Yeast two-hybrid systems are powerful proteomics tools for the discovery of protein-protein interactions. However, these systems are typically unable to detect interactions dependent on post-translational modifications such as tyrosine phosphorylation. We report a novel yeast tribrid system that expresses a potentially universal protein tyrosine kinase (PTK) substrate to detect diverse PTKs. Validation with the oncogenic kinases v-Abl and v-Src, which exhibit divergent substrate specificities, demonstrated significant potential for cloning PTKs en masse from cDNA libraries.     

Production of serpins using baculovirus expression systems

Headpin is a novel serine proteinase inhibitor (serpin) that is downregulated in many established HNSCC tumor cell lines and human oral SCC specimens. The use of the bacterial and yeast expression systems for headpin resulted in poor yields and proteins with low inhibitory activity. To circumvent these problems, we have developed a baculovirus-insect cell system for high-yield expression as well as fully functional protein. Here, we describe the strategies and methods used to express headpin in an insect cell heterologous system. In addition, procedures to purify the recombinant proteins are described. A metal affinity column followed by a gel-filtration chromatography provides a rapid and efficient method for large quantity preparation of headpin. This method should be useful as an alternative expression system for those serpins that are not purifiable when expressed using the Escherichia coli or yeast expression system.     

Structural overview of toxin–antitoxin systems in infectious bacteria: A target for developing antimicrobial agents

The bacterial toxin–antitoxin (TA) system is a module that may play a role in cell survival under stress conditions. Generally, toxin molecules act as negative regulators in cell survival and antitoxin molecules as positive regulators. Thus, the expression levels and interactions between toxins and antitoxins should be systematically harmonized so that bacteria can escape such harmful conditions. Since TA systems are able to control the fate of bacteria, they are considered potent targets for the development of new antimicrobial agents. TA systems are widely prevalent with a variety of systems existing in bacteria: there are three types of bacterial TA systems depending on the property of the antitoxin which binds either the protein toxin or mRNA coding the toxin protein. Moreover, the multiplicity of TA genes has been observed even in species of bacteria. Therefore, knowledge on TA systems such as the individual characteristics of TA systems, integrative working mechanisms of various TA systems in bacteria, interactions between toxin molecules and cellular targets, and so on is currently limited due to their complexity. In this regard, it would be helpful to know the structural characteristics of TA modules for understanding TA systems in bacteria. Until now, 85 out of the total structures deposited in PDB have been bacterial TA system proteins including TA complexes or isolated toxins/antitoxins. Here, we summarized the structural information of TA systems and analyzed the structural characteristics of known TA modules from several bacteria, especially focusing on the TA modules of several infectious bacteria.     

Counter-selectable marker for bacterial-based interaction trap systems

Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two different URA3 reporter systems were developed that allow robust counter-selection: (i) a single copy F' episome reporter and (ii) a co-cistronic HIS3-URA3 reporter vector. The HIS3-URA3 reporter can be used for either positive or negative selections in appropriate bacterial strains. These reagents extend the utility of the bacterial two-hybrid system as an alternative to its yeast-based counterpart.     

Bacterial and eukaryotic systems collide in the three Rs of Methanococcus

Methanococcus maripaludis S2 is a methanogenic archaeon with a well-developed genetic system. Its mesophilic nature offers a simple system in which to perform complementation using bacterial and eukaryotic genes. Although information-processing systems in archaea are generally more similar to those in eukaryotes than those in bacteria, the order Methanococcales has a unique complement of DNA replication proteins, with multiple MCM (minichromosome maintenance) proteins and no obvious originbinding protein. A search for homologues of recombination and repair proteins in M. maripaludis has revealed a mixture of bacterial, eukaryotic and some archaeal-specific homologues. Some repair pathways appear to be completely absent, but it is possible that archaeal-specific proteins could carry out these functions. The replication, recombination and repair systems in M. maripaludis are an interesting mixture of eukaryotic and bacterial homologues and could provide a system for uncovering novel interactions between proteins from different domains of life.     

Towards systems metabolic engineering in Pichia pastoris

The methylotrophic yeast Pichia pastoris is firmly established as a host for the production of recombinant proteins, frequently outperforming other heterologous hosts. Already, a sizeable amount of systems biology knowledge has been acquired for this non-conventional yeast. By applying various omics-technologies, productivity features have been thoroughly analyzed and optimized via genetic engineering. However, challenging clonal variability, limited vector repertoire and insufficient genome annotation have hampered further developments. Yet, in the last few years a reinvigorated effort to establish P. pastoris as a host for both protein and metabolite production is visible. A variety of compounds from terpenoids to polyketides have been synthesized, often exceeding the productivity of other microbial systems. The clonal variability was systematically investigated and strategies formulated to circumvent untargeted events, thereby streamlining the screening procedure. Promoters with novel regulatory properties were discovered or engineered from existing ones. The genetic tractability was increased via the transfer of popular manipulation and assembly techniques, as well as the creation of new ones. A second generation of sequencing projects culminated in the creation of the second best functionally annotated yeast genome. In combination with landmark physiological insights and increased output of omics-data, a good basis for the creation of refined genome-scale metabolic models was created. The first application of model-based metabolic engineering in P. pastoris showcased the potential of this approach. Recent efforts to establish yeast peroxisomes for compartmentalized metabolite synthesis appear to fit ideally with the well-studied high capacity peroxisomal machinery of P. pastoris. Here, these recent developments are collected and reviewed with the aim of supporting the establishment of systems metabolic engineering in P. pastoris.