Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.     

A human cDNA library for high-throughput protein expression screening

We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.     

An Automated High-Throughput Cell-Based Multiplexed Flow Cytometry Assay to Identify Novel Compounds to Target Candida albicans Virulence-Related Proteins

Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new antifungal drugs targeted to fungal virulence proteins using C. albicans as a model fungal pathogen.     

A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments.

A simple and sensitive radioimmunoassay using E. coli β-galactosidase as a model protein has been developed for the detection of specific translation products of foreign gene fragments cloned into plasmid or phage vectors. This immunoassay is based upon the coupling to an insoluble matrix of F(ab)′₂ fragments derived from the specific antiserum by pepsin digestion. The in situ analysis of phage plaques or of bacterial colonies is performed by overlaying the phage plaques or lysed bacterial colonies with a cellulose filter to which F(ab)′₂ fragments have been chemically coupled. The antigen bound to the filter is detected by subsequent incubations with undigested antiserum and with ¹²⁵I-labeled Staphylococcus aureus protein A followed by autoradiography. By coupling the F(ab)′₂ fragments to the wells of a plastic microtiter plate, liquid cultures can be analyzed quantitatively for the presence of antigen, making possible the analysis of heterogeneous cultures by sib selection. The detection threshold of the microtiter plate assay for liquid culture is shown to be <2 × 10⁸ molecules, or about 1 molecule of β-galactosidase per cell. The in situ immunoassay for bacterial colonies, which permits examination of about 1000 clones per plate, can easily detect microcolonies producing about 10 molecules of β-galactosidase per cell, while the in situ phage plaque assay, also capable of screening about 1000 plaques per plate, is even more sensitive, detecting <1 × 10⁷ molecules per bacteriophage plaque.     

Robust and Systematic Drug Screening Method Using Chemical Arrays and the Protein Library: Identification of Novel Inhibitors of Carbonic Anhydrase II

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.     

Robust and systematic drug screening method using chemical arrays and the protein library: identification of novel inhibitors of carbonic anhydrase II

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.     

A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species

Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.     

Oxidative stress-resistance assay for screening yeast strains overproducing heterologous proteins

Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast. In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would make superior strains producing heterologous proteins.     

Development of a high-throughput screening system for the compounds that inhibit collagen–protein interactions

Collagen-binding proteins (CBPs) play important roles in various physiological events. Some CBPs are regarded as targets for drug development; for example, platelet glycoprotein VI (GPVI) and heat shock protein 47 (HSP47) are promising targets for the development of novel antiplatelet and antifibrotic drugs, respectively. However, no systematic screening method to search compounds that inhibit collagen–CBP interactions have been developed, and only a few CBP inhibitors have been reported to date. In this study, a facile turbidimetric multiwell plate assay was developed to evaluate inhibitors of CBPs. The assay is based on the finding that CBPs retard spontaneous collagen fibril formation in vitro and that fibril formation is restored in the presence of compounds that interfere with the collagen–CBP interactions. Using the same platform, the assay was performed in various combinations of fibril-forming collagen types and CBPs. This homogeneous assay is simple, convenient, and suitable as an automated high-throughput screening system.     

A high-throughput approach to developing and optimizing mixed-mode membrane chromatography for protein purification

Membrane chromatography has been established as a viable alternative to packed-bed column chromatography for the purification of therapeutic proteins. Purification via membrane chromatography offers key advantages, including higher productivity and reduced buffer usage. Unlike column chromatography purification, the utilization of high-throughput screening in order to reduce development times and material requirements has been a challenge for membrane chromatography. This research focused on the development of a new, high-throughput screening technique for use in screening membrane chromatography conditions for monoclonal antibody purification. The developed screen utilizes a 96-well plate format, thereby allowing for the screening of multiple different membrane conditions at once. For this study, four mixed-mode cation exchange membranes and one cation exchange membrane were evaluated on the plate. The screen is performed in a similar manner to that of a resin slurry plate screen, however, instead of a single loading step, the antibody feed was loaded in 50 mg/ml increments up to a maximum loading of 450 mg/ml. Performing a similar, incremental loading on a resin plate would be impractical, as mixing times are substantially longer due to pore diffusion limitations. However, due to the significantly faster rate of mass transfer for membranes relative to resin, mixing times could be reduced by up to a factor of sixty on the membrane plate. Additional optimization showed that higher hydrophobicity can potentially lead to slower kinetics and mixing times that may need to be adjusted accordingly. The end result is a screen that has been proven to provide results comparable to those obtained on larger-scale membrane purification runs while also enabling exploration of a much greater operating space and significantly reducing the feed materials required.     

Real-time assay for monitoring membrane association of lipid-binding domains

The C2 domain is a common protein module which mediates calcium-dependent phospholipid binding. Several assays have previously been developed to measure membrane association. However, these assays either have technical drawbacks or are laborious to carry out. We now present a simple solution-based turbidity method for rapidly assaying membrane association of single lipid-binding domains in real time. We used the first C2 domain of synaptotagmin1 (C2A) as a model lipid-binding moiety. Our use of the common dimeric glutathione-S-transferase (GST) fusion tag allowed two C2A domains to be brought into close proximity. Consequently, calcium-triggered phospholipid binding by this artificially dimerized C2A resulted in liposomal aggregation, easily assayed by following absorbance of the solution at 350 nm. The assay is simple and sensitive and can be scaled up conveniently for use in a multiwell plate format, allowing high-throughput screening. In our screens, we identified nickel as a novel activator of synaptotagmin1 C2A domain membrane association. Finally, we show that the turbidity method can be applied to the study of other GST-tagged lipid-binding proteins such as epsin, protein kinase C-β, and synaptobrevin.     

Large-scale functional analysis using peptide or protein arrays

The array format for analyzing peptide and protein function offers an attractive experimental alternative to traditional library screens. Powerful new approaches have recently been described, ranging from synthetic peptide arrays to whole proteins expressed in living cells. Comprehensive sets of purified peptides and proteins permit high-throughput screening for discrete biochemical properties, whereas formats involving living cells facilitate large-scale genetic screening for novel biological activities. In the past year, three major genome-scale studies using yeast as a model organism have investigated different aspects of protein function, including biochemical activities, gene disruption phenotypes, and protein-protein interactions. Such studies show that protein arrays can be used to examine in parallel the functions of thousands of proteins previously known only by their DNA sequence.     

Development of phosphocellulose paper-based screening of inhibitors of lipid kinases: Case study with PI3Kβ

The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate the cellular signal transduction pathways involved in cell growth, proliferation, survival, apoptosis, and adhesion. Deregulation of these pathways are common in oncogenesis, and they are known to be altered in other metabolic disorders as well. Despite its huge potential as an attractive target in these diseases, there is an unmet need for the development of a successful inhibitor. Unlike protein kinase inhibitors, screening for lipid kinase inhibitors has been challenging. Here we report, for the first time, the development of a radioactive lipid kinase screening platform using a phosphocellulose plate that involves transfer of radiolabeled [γ-³²P]ATP to phosphatidylinositol 4,5-phosphate forming phosphatidylinositol 3,4,5-phosphate, captured on the phosphocellulose plate. Enzyme kinetics and inhibitory properties were established in the plate format using standard inhibitors, such as LY294002, TGX-221, and wortmannin, having different potencies toward PI3K isoforms. ATP and lipid apparent K_m for both were determined and IC₅₀ values generated that matched the historical data. Here we report the use of a phosphocellulose plate for a lipid kinase assay (PI3Kβ as the target) as an excellent platform for the identification of novel chemical entities in PI3K drug discovery.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.     

Solid-phase assays for small molecule screening using sol-gel entrapped proteins.

With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput screening of small molecule libraries and mixtures.     

Screening for ligands using a generic and high-throughput light-scattering-based assay

Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.     

High-throughput protein expression screening and purification in Escherichia coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, to obtain milligrams of soluble proteins is still challenging since many proteins are expressed in an insoluble form without optimization. Therefore when working with tens of proteins or protein domains it is recommended that high-throughput expression screening at a small scale (1-4ml of culture) is carried out to identify the optimal conditions for soluble protein production. Once determined, these culture conditions can be applied at a large scale to produce sufficient protein for structural or functional studies. We describe a procedure that has enabled the systematic screening of culture conditions or fusion-tags on hundreds of cultures per week. The analysis of the optimal conditions for the soluble production of these proteins helped us to design a simple and efficient protocol for soluble protein expression screening. This protocol has since been used on hundreds of proteins and is illustrated with the genome wide scale production of proteins containing the DNA binding domains of Ciona intestinalis.     

A versatile high-throughput screen for inhibitors of lipid kinase activity: development of an immobilized phospholipid plate assay for phosphoinositide 3-kinase gamma

The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K gamma isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening. The authors report the development of a simple and robust 384-well plate assay that is based on(33) P-phosphate transfer from radiolabeled [gamma(33) P]ATP to phosphatidylinositol immobilized on Maxisorp plates. The established assay format for PI3K gamma was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K gamma determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K gamma was observed at pH 7.0. The apparent K(m) value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3 microM (6.0-8.6 microM, 95% confidence interval [CI]). IC(50) values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.