Overexpression of membrane proteins from higher eukaryotes in yeasts

Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug–target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.     

Assays for monitoring viral manipulation of host ARE-mRNA turnover

Early host responses to viral infection rapidly induce an antiviral gene expression program that limits viral replication and recruits sentinel cells of the innate immune system. These responses are mediated by cytokines. The mRNAs that encode cytokines typically harbor destabilizing adenine- and uridine-rich elements (AREs) that direct their constitutive degradation in the cytoplasm. In response to a variety of signals, including viral infection, small pools of cytoplasmic ARE-mRNAs are rapidly stabilized and translated. Thus, mRNA stability plays a key role in antiviral gene expression. Intriguingly, recent studies have identified viral proteins that specifically target ARE-mRNAs for stabilization, suggesting that certain proteins encoded by ARE-mRNAs may be advantageous for infection. Here, we discuss the development of a suite of sensitive and complementary assays to monitor ARE-mRNA turnover. These include luciferase- and destabilized-GFP-based assays that can be adapted for high-throughput screening applications.     

Mammalian expression cloning of nucleic acid binding proteins by agarose thin-layer gelshift clone selection

Here we report a functional screening technique to identify cDNAs encoding mammalian nucleic acid binding proteins. We have combined cDNA expression cloning with the agarose thin-layer gelshift assay technique to detect specific nucleic acid binding proteins from a mammalian expression library. We divided this cDNA expression library into multiple pools and transfected mammalian cells with the individual pools. Following transfection, we tested the expressed proteins for DNA-binding activity by agarose thin-layer electrophoretic gelshift assay. After we identified a single expression poolfor the presence of a DNA-binding protein, the corresponding cDNA pool was further divided into smaller aliquots. Then, the cDNA expression and gelshift clone selection was repeated until a single clone was isolated In contrast to traditional polyacrylamide gels, the agarose thin-layer is significantly faster and resolves larger DNA-protein complexes. This method can be widely used for the cDNA cloning of DNA- and RNA-binding proteins from various mammalian host cells.     

Cell-Surface display of heterologous proteins: From high-throughput screening to environmental applications

A variety of expression systems for the display of either short peptides or fully folded proteins on E.coli and, to a lesser extent, on Gram-positive bacteria have been developed. The expression of proteins on the surface of microbial cells has proved extremely important for numerous applications ranging from combinatorial library screening and protein engineering, to whole cell biocatalysts and adsorbants for bioremediation purposes.     

微生物表面展示技术及其在环境污染治理中的应用

微生物表面展示技术是通过基因工程手段,将短的外源肽或蛋白质表达在微生物细胞表面,该技术可以应用于开发活的细菌疫苗、筛选抗体库、生产生物细胞吸附剂以及制备整细胞生物催化剂。通过金属高效结合肽的肽库筛选和微生物展示技术,将金属结合肽直接展示在微生物的表面,用于处理环境中的重金属污染,为环境中重金属污染的防治提供了一条崭新的途径。利用微生物表面展示技术制备整细胞催化剂,用于有毒有机污染物的处理,可以极大地加快污染物的降解速率。简要介绍了微生物表面展示技术及其在重金属污染治理和毒性有机污染物的脱毒等环境生物修复方面的最新研究进展。     

Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using ‘MAX’ randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.     

Lung NF-kappaB activation and neutrophil recruitment require IL-1 and TNF receptor signaling during pneumococcal pneumonia

Pulmonary inflammation is an essential component of the host defense against Streptococcus pneumoniae infection of the lungs. The early response cytokines, TNF-alpha and IL-1, are rapidly induced upon microbial exposure. Mice deficient in all TNF- and IL-1-dependent signaling receptors were used to determine the roles of these cytokines during pneumococcal pneumonia. The deficiency of signaling receptors for TNF and IL-1 decreased bacterial clearance. Neutrophil recruitment to alveolar air spaces was impaired by receptor deficiency, as was pulmonary expression of the neutrophil chemokines KC and MIP-2. Because NF-kappaB mediates the expression of both chemokines, we assessed NF-kappaB activation in the lungs. During pneumococcal pneumonia, NF-kappaB proteins translocate to the nucleus and activate gene expression; these functions were largely abrogated by the deficiency of receptors for TNF-alpha and IL-1. Thus, the combined deficiency of TNF and IL-1 signaling reduces innate immune responses to S. pneumoniae in the lungs, probably due to essential roles for these receptors in activating NF-kappaB.     

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.     

An entirely cell-based system to generate single-chain antibodies against cell surface receptors

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.     

Development of a method for screening short-lived proteins using green fluorescent protein

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.     

Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.     

MicroRNAs as mediators of insect host–pathogen interactions and immunity

Insects are the most successful group of animals on earth, owing this partly to their very effective immune responses to microbial invasion. These responses mainly include cellular and humoral responses as well as RNA interference (RNAi). Small non-coding RNAs (snRNAs) produced through RNAi are important molecules in the regulation of gene expression in almost all living organisms; contributing to important processes such as development, differentiation, immunity as well as host–microorganism interactions. The main snRNAs produced by the RNAi response include short interfering RNAs, microRNAs and piwi-interacting RNAs. In addition to the host snRNAs, some microorganisms encode snRNAs that affect the dynamics of host–pathogen interactions. In this review, we will discuss the latest developments in regards to the role of microRNA in insect host–pathogen interactions and provide some insights into this rapidly developing area of research.     

A novel third chromosomal locus controls susceptibility to Autographa californica multiple nucleopolyhedrovirus in the silkworm,Bombyx mori

Baculovirus demonstrates specific infection spectrums and thus one certain host exhibits particular response to single baculovirus isolate. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is considered to be not an innate pathogen to Bombyx mori, but some silkworm strains have been identified to be permissive to AcMNPV, indicating the positive or negative involvement of certain host factors in baculovirus replications in vivo. To provide a fundamental knowledge of this process, we performed large-scale screening to investigate the responses of 448 silkworm strains against recombinant AcMNPV inoculation. By genetic analysis between permissive and resistant strains identified, we further confirmed that a potential corresponding locus on chromosome 3 regulates host responses to AcMNPV in silkworm. Additionally, we found that it is available for AcMNPV–silkworm baculovirus expression vector system to produce proteins of interest.     

Cell‐free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite‐related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell‐free protein synthesis system for large‐scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high‐throughput expression and identification of individual genes comprising the library, a cell‐free synthesis reaction was designed such that both the template DNA and the expressed proteins were co‐immobilized on the same microbeads, leading to microbead‐based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832–837, 2017     

Expression of maize prolamins in Escherichia coli

We have constructed a cDNA expression library of developing corn (Zea mays L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250–900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with ³²P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed.     

Tapping diversity lost in transformations--in vitro amplification of ligation reactions

Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>10(9) transformants in Escherichia coli and 10(5)-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.