High-throughput Protein Expression Research Articles

High-Throughput Protein Expression Screening of Cell-Surface Protein Ectodomains.

Cell-surface receptors can be difficult to express and purify for structural and biochemical studies due to low expression levels, misfolding, aggregation, and instability. Cell-surface receptor ectodomains are more amenable to large-scale production, but this requires designing and testing various truncation constructs. However, since each protein is unique, testing these constructs individually for many targets is a time-consuming process. In this context, we present a high-throughput ELISA fluorescence approach that allows the rapid assessment of numerous recombinant constructs simultaneously. Cell-surface ectodomains are expressed in small scale, enzymatically biotinylated, and detected using a C-terminal His-tag. As an example, we tested the expression of truncation constructs for the neurexin, neuroligin, and latrophilin families and show that the small-scale ELISA allowed us to prioritize well-expressing construct for large-scale production. By employing this method, one can efficiently detect clones with low expression levels, streamlining the process and saving valuable time in identifying optimal candidates for further study.

Plant-based biopharmaceutical engineering.

Plants can be engineered to recombinantly produce high-quality proteins such as therapeutic proteins and vaccines, also known as molecular farming. Molecular farming can be established in various settings withminimal cold-chain requirements and could thus ensure rapid and global-scale deployment of biopharmaceuticals, promoting equitable access to pharmaceuticals. State of the artplant-based engineering relies on rationally assembled genetic circuits, engineered to enable the high-throughput and rapid expression of multimeric proteins with complex post-translational modifications. In this Review, we discuss the design of expression hosts and vectors, including Nicotiana benthamiana, viral elements and transient expression vectors, for the production of biopharmaceuticals in plants. We examineengineering ofpost-translational modifications and highlight the plant-based expression of monoclonalantibodies and nanoparticles, such as virus-like particles and protein bodies. Techno-economic analyses suggest a cost advantage of molecular farming compared with mammalian cell-based protein production systems. However, regulatory challenges remain to be addressed to enable the widespread translation of plant-based biopharmaceuticals.

ESKAPE Act Plus: Pathway Activation Analysis for Bacterial Pathogens.

The last 20 years have witnessed an explosion in publicly available gene expression and proteomic data and new tools to help researchers analyze these data. Tools typically include statistical approaches to identify differential expression, integrate prior knowledge, visualize results, and suggest how differential expression relates to changes in phenotype. Here, we provide a simple web-based tool that bridges some of the gaps between the functionality available to those studying eukaryotes and those studying prokaryotes. Specifically, our Shiny web application ESKAPE Act PLUS allows researchers to upload results of high-throughput bacterial gene or protein expression experiments from 13 species, including the six ESKAPE pathogens, to our system and receive (i) an analysis of which KEGG pathways or GO terms are significantly activated or repressed, (ii) visual representations of the magnitude of activation or repression in each category, and (iii) detailed diagrams showing known relationships between genes in each regulated KEGG pathway and fold changes of individual genes. Importantly, our statistical approach does not require users to identify which genes or proteins are differentially expressed. ESKAPE Act PLUS provides high-quality statistics and graphical representations not available using other web-based systems to assess whether prokaryotic biological functions are activated or repressed by experimental conditions. To our knowledge, ESKAPE Act PLUS is the first application that provides pathway activation analysis and pathway-level visualization of gene or protein expression for prokaryotes. IMPORTANCE ESKAPE pathogens are bacteria of concern because they develop antibiotic resistance and can cause life-threatening infections, particularly in more susceptible immunocompromised people. ESKAPE Act PLUS is a user-friendly web application that will advance research on ESKAPE and other pathogens commonly studied by the biomedical community by allowing scientists to infer biological phenotypes from the results from high-throughput bacterial gene or protein expression experiments. ESKAPE Act PLUS currently supports analysis of 23 strains of bacteria from 13 species and can also be used to re-analyze publicly available data to generate new findings and hypotheses for follow-up experiments.

Identifying Leptospira interrogans putative virulence factors with a yeast protein expression screen.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: • High-throughput cloning and expression of leptospiral proteins in yeast. • Heterologous expression of nine leptospiral proteins inhibited yeast growth. • An uncharacterized protein LIC10280 maybe a putative VF for further validation.

Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray.

The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro transcription/translation (IVTT) systems-a similarly high-throughput protein expression method-are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on the same protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from different expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clear correlation between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.

Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart

The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in the heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. We developed an integrated process for simultaneous isolation and culture of the main cell types comprising the microcirculation in adult mouse heart: endothelial cells, pericytes, and vascular smooth muscle cells. These cell types were characterized with isobaric labeling quantitative proteomics and mRNA sequencing. We defined microvascular cell proteomes, identified novel protein markers, and confirmed established cell-specific markers. Our results allow identification of unique markers and regulatory proteins that govern microvascular physiology and pathology.

Better Agreement of Human Transcriptomic and Proteomic Cancer Expression Data at the Molecular Pathway Activation Level.

Previously, we have shown that the aggregation of RNA-level gene expression profiles into quantitative molecular pathway activation metrics results in lesser batch effects and better agreement between different experimental platforms. Here, we investigate whether pathway level of data analysis provides any advantage when comparing transcriptomic and proteomic data. We compare the paired proteomic and transcriptomic gene expression and pathway activation profiles obtained for the same human cancer biosamples in The Cancer Genome Atlas (TCGA) and the NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) projects, for a total of 755 samples of glioblastoma, breast, liver, lung, ovarian, pancreatic, and uterine cancers. In a CPTAC assay, expression levels of 15,112 protein-coding genes were profiled using the Thermo QE series of mass spectrometers. In TCGA, RNA expression levels of the same genes were obtained using the Illumina HiSeq 4000 engine for the same biosamples. At the gene level, absolute gene expression values are compared, whereas pathway-grade comparisons are made between the pathway activation levels (PALs) calculated using average sample-normalized transcriptomic and proteomic profiles. We observed remarkably different average correlations between the primary RNA- and protein expression data for different cancer types: Spearman Rho between 0.017 (p = 1.7 × 10−13) and 0.27 (p < 2.2 × 10−16). However, at the pathway level we detected overall statistically significantly higher correlations: averaged Rho between 0.022 (p < 2.2 × 10−16) and 0.56 (p < 2.2 × 10−16). Thus, we conclude that data analysis at the PAL-level yields results of a greater similarity when comparing high-throughput RNA and protein expression profiles.

Mining Protein Expression Databases Using Network Meta-Analysis.

Public databases featuring original, raw data from "Omics" experiments enable researchers to perform meta-analyses by combining either the raw data or the summarized results of several independent studies. In proteomics, high-throughput protein expression data is measured by diverse techniques such as mass spectrometry, 2-D gel electrophoresis or protein arrays yielding data of different scales. Therefore, direct data merging can be problematic, and combining the summarized data of the individual studies can be advantageous. A special form of meta-analysis is network meta-analysis, where studies with different settings of experimental groups can be combined. However, all studies must be linked by one experimental group that has to appear in each study. Usually that is the control group. Then, a study network is formed and indirect statistical inferences can also be made between study groups that appear not in each of the studies.In this chapter, we describe the working principle of and available software for network meta-analysis. The applicability to high-throughput protein expression data is demonstrated in an example from breast cancer research. We also describe the special challenges when applying this method.

Protein Microarrays for Ocular Diseases.

The eye is a multifaceted organ organized in several compartments with particular properties that reflect their diverse functions. The prevalence of ocular diseases is increasing, mainly because of its relationship with aging and of generalized lifestyle changes. However, the pathogenic molecular mechanisms of many common eye pathologies remain poorly understood. Considering the unquestionable importance of proteins in cellular processes and disease progression, proteomic techniques, such as protein microarrays, represent a valuable approach to analyze pathophysiological protein changes in the ocular environment. This technology enables to perform multiplex high-throughput protein expression profiling with minimal sample volume requirements broadening our knowledge of ocular proteome network in eye diseases.In this review, we present a brief summary of the main types of protein microarrays (antibody microarrays, reverse-phase protein microarrays, and protein microarrays) and their application for protein change detection in chronic ocular diseases such as dry eye, age-related macular degeneration, diabetic retinopathy, and glaucoma. The validation of these specific protein changes in eye pathologies may lead to the identification of new biomarkers, depiction of ocular disease pathways, and assistance in the diagnosis, prognosis, and development of new therapeutic options for eye pathologies.

Construction of gateway-compatible baculovirus expression vectors for high-throughput protein expression and in vivo microcrystal screening

Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies.

Screening Intrinsically Disordered Regions for Short Linear Binding Motifs.

The intrinsically disordered regions of the proteome are enriched in short linear motifs (SLiMs) that serve as binding sites for peptide binding proteins. These interactions are often of low-to-mid micromolar affinities and are challenging to screen for experimentally. However, a range of dedicated methods have been developed recently, which open for screening of SLiM-based interactions on large scale. A variant of phage display, termed proteomic peptide phage display (ProP-PD), has proven particularly useful for the purpose. Here, we describe a complete high-throughput ProP-PD protocol for screening intrinsically disordered regions for SLiMs. The protocol requires some basic bioinformatics skills for the design of the library and for data analysis but can be performed in a standard biochemistry lab. The protocol starts from the construction of a library, followed by the high-throughput expression and purification of bait proteins, the phage selection, and the analysis of the binding-enriched phage pools using next-generation sequencing. As the protocol generates rather large data sets, we also emphasize the importance of data management and storage.

Selective androgen receptor modulators (SARMs) have specific impacts on the mouse uterus.

Selective androgen receptor modulators (SARMs) have been proposed as therapeutics for women suffering from breast cancer, muscle wasting or urinary incontinence. The androgen receptor (AR) is expressed in the uterus but the impact of SARMs on the function of this organ is unknown. We used a mouse model to compare the impact of SARMs (GTx-007/Andarine®, GTx-024/Enobosarm®), Danazol (a synthetic androstane steroid) and dihydrotestosterone (DHT) on tissue architecture, cell proliferation and gene expression. Ovariectomised mice were treated daily for 7 days with compound or vehicle control (VC). Uterine morphometric characteristics were quantified using high-throughput image analysis (StrataQuest; TissueGnostics), protein and gene expression were evaluated by immunohistochemistry and RT-qPCR, respectively. Treatment with GTx-024, Danazol or DHT induced significant increases in body weight, uterine weight and the surface area of the endometrial stromal and epithelial compartments compared to VC. Treatment with GTx-007 had no impact on these parameters. GTx-024, Danazol and DHT all significantly increased the percentage of Ki67-positive cells in the stroma, but only GTx-024 had an impact on epithelial cell proliferation. GTx-007 significantly increased uterine expression of Wnt4 and Wnt7a, whereas GTx-024 and Danazol decreased their expression. In summary, the impact of GTx-024 and Danazol on uterine cells mirrored that of DHT, whereas GTx-007 had minimal impact on the tested parameters. This study has identified endpoints that have revealed differences in the effects of SARMs on uterine tissue and provides a template for preclinical studies comparing the impact of compounds targeting the AR on endometrial function.

A High-Throughput Platform for the Generation of Synthetic Ab Clones by Single-Strand Site-Directed Mutagenesis.

Current developments in meta-data analysis and predictive computational models offer alternative routes for the identification of antibodies. In silico-based technologies and NGS data analysis from Ab phage-display selections offer expanded selections of Ab candidates. Accordingly, the identified de novo Abs with predicted selectivity for a target antigen must undergo rapid gene synthesis for downstream Ab characterizations. Here we describe a high-throughput strategy for the generation of synthetic Ab clones for expression as Fab proteins in Escherichia coli. Our approach utilizes simultaneous single-stranded site-directed mutagenesis of diversified Ab regions of a phagemid template with engineered complementary determining regions that contain multiple stop codon and restriction enzyme sites. Subsequently, we perform rapid screening of Ab DNA clones for correct gene assemblies by high-throughput Ab-phage protein expression screens. Identified sequences are corroborated by Sanger DNA sequencing analysis. In summary, our work describes a rapid and cost-effective platform for the high-throughput synthesis of synthetic Ab genes as Fab proteins for implementation into downstream proteinvalidation pipelines.

High-Throughput Protein Analysis Using Negative Stain Electron Microscopy and 2D Classification.

High-throughput protein expression and purification allows for fast triaging of several constructs based on expression levels, protein integrity, and solubility. While this technology has been successfully adopted to prioritize constructs for structural biology, it could not inform on important biochemical properties such as domain architecture, homogeneity, and flexibility. Negative staining electron microscopy can be used to quickly evaluate these properties and, if coupled to single particle analysis, can inform on the architecture and conformational state of nearly any protein sample. Here we describe a protocol for negative stain sample preparation, imaging, and two-dimensional (2D) data analysis applicable to a variety of protein complexes. We discuss in more detail a specific application of this technology to large molecule studies to determine the binding sites of individual antibodies on target antigens.

High-Throughput Protein Production in Yeast.

Yeasts are versatile single-celled fungi that grow to high cell densities on inexpensive media. With well-studied genetics and metabolism and a wealth of knowledge available about their propagation and growth in academic as well as industrial settings, yeasts have long been used for recombinant protein production of isolated proteins and multisubunit complexes. They can be easily adapted to high-throughput protein expression pipelines. Importantly, the outcome from small-scale expression evaluations in high-throughput mode is scalable to laboratory and industrial scales using well-established procedures. In this chapter, we offer a state-of-the-art perspective on currently available high-throughput pipelines for protein production in S. cerevisiae and P. pastoris and discuss future challenges and avenues for improvement.

Screening and validating the prognostic biomarkers of hepatocelluar carcinoma based on reverse phase protein arrays

Objective To screen and validate the prognostic biomarkers of patients with hepatocelluar carcinoma (HCC) based on reverse phase protein array (RPPA). Methods All public RPPA data (HCC patients) in TCGA database before December 2017 were enrolled in this study. Univariate and multivariate Cox analysis were used to screen the target proteins with prognostic value. From January 2013 to January 2014, 121 HCC patients who were treated in the Affiliated Hospital of North Sichuan Medical College were enrolled in the study. Prognostic related proteins screened from RPPA data were detected in these patients’ tumor specimen. Then, the correlations between the expression of the screened protein and prognosis was further analyzed. Results In the 218 protein, 23 was related with HCC patient prognosis. Multivariate Cox regression analysis showed that high expression of NOTCH1 (HR=1.515, 95% CI: 1.287~1.845, P 0.05). Conclusion RPPA, as a high throughput protein expression quantitative technique, can be used for screening prognostic markers, and the reliability of the screening results is higher in general. Key words: Hepatocellular carcinoma; Prognosis; Biomarker; Reverse phase protein arrays

New tools for high-throughput expression of fungal secretory proteins in Saccharomyces cerevisiae and Pichia pastoris.

SummaryHeterologous protein expression in yeast, mostly in Saccharomyces cerevisiae and Pichia pastoris, is a well‐established and widely used technique. It typically requires the construction of an expression vector in Escherichia coli containing the foreign gene and its subsequent transformation into yeast. Although simple, this procedure has important limitations for the expression of large numbers of proteins, that is, for the generation of protein libraries. We describe here the development of a novel system for the easy and fast expression of heterologous proteins both in S. cerevisiae and in P. pastoris, under the control of the GAL1 and AOX1 promoters respectively. Expression in S. cerevisiae requires only the transformation of yeast cells with an unpurified PCR product carrying the gene to be expressed, and the expression of the same gene in P. pastoris requires only the isolation of the plasmid generated in S. cerevisiae and its transformation into this second yeast, thus making this system suitable for high‐throughput projects. The system has been tested by the extracellular expression of 30 secretory fungal proteins.

Mammalian cell culture density determination using a laser through-beam sensor.

High-throughput protein expression platforms are increasingly used to produce proteins for many applications: to support studies in structure/function, regulation and proteomics, as well as for direct use as potential biotherapeutic agents for medical applications. Here we describe a device that we refer to as the flask density reader (FDR) consisting of a through-beam laser and sensor, and a customized culture flask-receiving nest. The FDR has been integrated onto GNF System™'s automated protein expression platform to enable rapid, noninvasive, fully automated spectrophotometric determination of cell densities in suspension mammalian cell cultures. The FDR reduces the risk of culture contamination from frequent flask sampling and greatly reduces the time and effort needed to count cells using off-line methods.

GEView (Gene Expression View) Tool for Intuitive and High Accessible Visualization of Expression Data for Non-Programmer Biologists

This article describes how the last decade has been characterized by the production of huge amounts of different types of biological data. Following that, a flood of bioinformatics tools have been published. However, many of these tools are commercial, or require computational skills. In addition, not all tools provide intuitive and highly accessible visualization of the results. The authors have developed GEView (Gene Expression View), which is a free, user-friendly tool harboring several existing algorithms and statistical methods for the analysis of high-throughput gene, microRNA or protein expression data. It can be used to perform basic analysis such as quality control, outlier detection, batch correction and differential expression analysis, through a single intuitive graphical user interface. GEView is unique in its simplicity and highly accessible visualization it provides. Together with its basic and intuitive functionality it allows Bio-Medical scientists with no computational skills to independently analyze and visualize high-throughput data produced in their own labs.

Microfluidic Transfection for High-Throughput Mammalian Protein Expression.

Mammalian synthetic biology and cell biology would greatly benefit from improved methods for highly parallel transfection, culturing, and interrogation of mammalian cells. Transfection is routinely performed on high-throughput microarrays, but this setup requires manual cell culturing and precludes precise control over the cell environment. As an alternative, microfluidic transfection devices streamline cell loading and culturing. Up to 280 transfections can be implemented on the chip at high efficiency. The culturing environment is tightly regulated and chambers physically separate the transfection reactions, preventing cross-contamination. Unlike typical biological assays that rely on end-point measurements, the microfluidic chip can be integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time.