High-throughput Selection Research Articles

Bacteriophage Vehicles for Phage Display: Biology, Mechanism, and Application

The phage display technique is a powerful tool for selection of various biological agents. This technique allows construction of large libraries from the antibody repertoire of different hosts and provides a fast and high-throughput selection method. Specific antibodies can be isolated based on distinctive characteristics from a library consisting of millions of members. These features made phage display technology preferred method for antibody selection and engineering. There are several phage display methods available and each has its unique merits and application. Selection of appropriate display technique requires basic knowledge of available methods and their mechanism. In this review, we describe different phage display techniques, available bacteriophage vehicles, and their mechanism.

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding compared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins’ aptamers, as well as two suspected background binding sequences, to eight targets running serially. The targets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device’s plate-based format with other complementary plate-based systems for all downstream biochemical processes and analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections. FigureSchematic breakdown of a microplate-based enrichment device for the selection of aptamers (MEDUSA), which can be customized and assembled in both parallel and serial configurations. Up to 96 selections can be performed simultaneously. Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7661-7) contains supplementary material, which is available to authorized users.

High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system

Therapeutic recombinant monoclonal antibodies (mAbs) are commonly produced by high-expressing, clonal, mammalian cells. Creation of these clones for manufacturing remains heavily reliant on stringent selection and gene amplification, which in turn can lead to genetic instability, variable expression, product heterogeneity and prolonged development timelines. Inclusion of cis-acting ubiquitous chromatin opening elements (UCOE™) in mammalian expression vectors has been shown to improve productivity and facilitate high-level gene expression irrespective of the chromosomal integration site without lengthy gene amplification protocols. In this study we have used high-throughput robotic clone selection in combination with UCOE™ containing expression vectors to develop a rapid, streamlined approach for early-stage cell line development and isolation of high-expressing clones for mAb production using Chinese hamster ovary (CHO) cells. Our results demonstrate that it is possible to go from transfection to stable clones in only 4 weeks, while achieving specific productivities exceeding 20 pg/cell/day. Furthermore, we have used this approach to quickly screen several process-crucial parameters including IgG subtype, enhancer-promoter combination and UCOE™ length. The use of UCOE™-containing vectors in combination with automated robotic selection provides a rapid method for the selection of stable, high-expressing clones.

Antibody Library Display on a Mammalian Virus Vector: Combining the Advantages of Both Phage and Yeast Display into One Technology

Utilizing a vaccinia virus based library technology, we previously developed an antibody discovery platform that enabled efficient selection of fully functional IgG antibodies from highly diverse immunoglobulin gene libraries expressed on the surface of mammalian cells. Recently, we have further modified this platform to enable efficient expression of a library of fully human antibodies on the surface of vaccinia virus; an enveloped mammalian virus. Similar in concept to phage display, conditions are utilized under which each vaccinia virion expresses a single antibody specificity on its surface. Various panning and magnetic bead based methods have been developed to allow screening of a library of vaccinia- MAb virions and selection of recombinant vaccinia virus encoding specific antibodies. Upon infection of mammalian cells the antibody is not only incorporated into newly produced virus, it is also displayed on the surface of the host cell. Similar to methods utilized in yeast display, the cells displaying vaccinia encoded antibody can also be selected using a combination of magnetic beads and cell sorting, and the virus encoding the specific antibody heavy and light chains readily recovered and analyzed. This technology allows for rapid high throughput selection of vaccinia-MAb virions in a cell free panning system, followed by cell based screening for high specificity and fine selection of optimal antibodies.

Linking genotype to phenotype on beads: high throughput selection of peptides with biological function

Although peptides are well recognised biological molecules in vivo, their selection from libraries is challenging because of relative low affinity whilst in linear conformation. We hypothesized that multiplexed peptides and DNA on the surface of beads would provide a platform for enhanced avidity and the selection of relevant peptides from a library (ORBIT bead display). Using human immunodeficiency virus (HIV-1) gp120 as a target, we identify peptides that inhibit HIV-1 replication in vitro through blocking of protein:protein interaction with the co-receptor CCR5. The bead display approach has many potential applications for probing biological systems and for drug lead development.

Programming adaptive control to evolve increased metabolite production

The complexity inherent in biological systems challenges efforts to rationally engineer novel phenotypes, especially those not amenable to high-throughput screens and selections. In nature, increased mutation rates generate diversity in a population that can lead to the evolution of new phenotypes. Here we construct an adaptive control system that increases the mutation rate in order to generate diversity in the population, and decreases the mutation rate as the concentration of a target metabolite increases. This system is called feedback-regulated evolution of phenotype (FREP), and is implemented with a sensor to gauge the concentration of a metabolite and an actuator to alter the mutation rate. To evolve certain novel traits that have no known natural sensors, we develop a framework to assemble synthetic transcription factors using metabolic enzymes and construct four different sensors that recognize isopentenyl diphosphate in bacteria and yeast. We verify FREP by evolving increased tyrosine and isoprenoid production.

Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool

Low expression, poor solubility, and polyspecificity are significant obstacles that have impeded the development of antibodies discovered from in vitro display libraries. Current biophysical characterization tools that identify these 'developability' problems are typically only applied after the discovery process, and thus limited to perhaps a few hundred candidates. We report a flow cytometric assay using a polyspecificity reagent (PSR) that allows for the identification and counter selection of polyspecific antibodies both during and after the selection process. The reported assay correlates well with cross-interaction chromatography, a surrogate for antibody solubility, as well as a baculovirus particle enzyme-linked immunosorbent assay, a surrogate for in vivo clearance. However, unlike these assays, PSR labeling is compatible both with screening of individual antibodies as well as selections of large antibody libraries. To this end, we demonstrate the ability to counter-select against polyspecificity while enriching for antigen affinity from a diverse antibody library, which enables simultaneous evolution of both antigen binding and superior non-target-related properties during the discovery process.

Enhanced Stem Cell Pluripotency in Surface‐Modified Electrospun Fibrous Matrices

A previously screened "hit chemistry" (N-[3-(dimethylamino)propyl] methacrylamide) that supports strong attachment and long-term self-renewal of ES cells is selected and grafted to poly(ether sulfone) (PES) fibrous matrices through plasma-induced graft polymerization. The 3D modified fibers exhibit higher cell proliferation and greater expression of pluripotency markers of mouse ES cells than 2D membranes. It is the first demonstration of scaling up an optimal synthetic surface chemistry in 2D using a high throughput synthesis, screening, and selection method to 3D that strongly influences pluripotent stem cell growth.

High throughput selection of co-stimulation molecules for 3rd generation designer T cells (P4362)

Abstract Ample co-stimulatory interactions between antigen-presenting cells and T cells are essential for an effective immune response. Similarly, co-stimulation related immuno-experiences have also been considered critical for the successful tumor eradication by T cells in adoptive immunotherapy cases of: conventional tumor infiltrating T cells for melanoma, chimeric antigen receptor (CAR)-expressing EBV-CTLs for EBV-associated malignancies and the recent CD19 CAR T cells for B-cell neoplasms. However, the specific co-stimulation signals playing the major role in successful immunotherapies are still not identified. Here, a series of constructs has been made to compare the effects of most known co-stimulation molecules, including 4-1BB, CD2, CD27, CD28, CD30, LFA1, GITR, HVEM, ICOS and OX40, in the 1st generation designer T cells bearing anti-CEA scFv-linked CD3ζ signal, and in the 2nd generation designer T cells with anti-CEA scFv linked with CD3ζ plus CD28 signal. Corresponding designer T cells’ function in vitro in proliferation/survival, cytotoxicity and cytokine secretion, and in vivo in animal models, will be tested and compared with the 1st/2nd generation designer T cells. The ones standing out will be delineated for their mechanisms from co-stimulation ligation to specific intracellular signal pathway that lead to the T cell phenotype responsible for final tumor rejection, and will be applied for 3rd generation designer T cell development.

Generation of monoclonal antibody-producing mammalian cell lines

Clinical effectiveness has driven the commercial success of monoclonal antibody (mAb) products. Mammalian cells are currently the preferred system for large-scale production as the mAbs produced are biochemically similar to human forms. The cell line generation process is tedious and time-consuming as clones with high productivity, stable long-term expression and good product quality are rare occurrences. Cell line generation efficiency and mAb quality can be improved through host cell engineering, vector optimization and high-throughput clone selection. Targeted integration into predetermined sites on the chromosome is a promising new area being explored. This review covers existing technology and recent progress made in improving various aspects of the stable cell line generation process for mAb production.

A cross-contamination-free SELEX platform for a multi-target selection strategy

Multi-target aptamer selection, known as multiplex systematic evolution of ligands by exponential enrichment (SELEX), is rapidly drawing interest because of its potential to enable high-speed, high-throughput aptamer selection. The parallelization of chemical processes by integrating microfluidic unit operations is a key strategy for developing a multiplex SELEX process. One of the potential problems with on-chip multiplexing chemical processes is cross-contamination. In order to avoid this, we propose a microfluidic network platform that uses pneumatic valves to allow the serial loading and incubation of aptamers with sol-gel entrapped target proteins. After target binding inside the sol-gels, the cross-contamination-free parallel elution of specifically bound aptamers is performed. The platform allows selective binding with five different targets immobilized in sol-gel spots. When eluting bound species, cross-contamination is avoided by sealing the adjacent elution chambers from each other using the pneumatic microvalves. Consequently, we demonstrate specific aptamer binding to the respective protein target and subsequent aptamer elution without any cross-contamination. This proof of concept opens the way to increased automation and microscale parallel processing of the SELEX methodology.

Synthesis of nanostructured and biofunctionalized water-in-oil droplets as tools for homing T cells.

Activation, ex vivo expansion of T cells, differentiation into a regulatory subset, and its phenotype-specific high-throughput selection represent major challenges in immunobiology. In part, this is due to the lack of technical means to synthesize suitable 3D extracellular systems to imitate ex vivo the cellular interactions between T cells and antigen-presenting cells (APCs). In this study, we synthesized a new type of gold-linked surfactant and used a drop-based microfluidic device to develop and characterize novel nanostructured and specifically biofunctionalized droplets of water-in-oil emulsions as 3D APC analogues. Combining flexible biofunctionalization with the pliable physical properties of the nanostructured droplets provided this system with superior properties in comparison with previously reported synthetic APC analogues.

Wide Screening of Phage-Displayed Libraries Identifies Immune Targets in Planta

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2×107 different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains.

BAPJ69-4A: A yeast two-hybrid strain for both positive and negative genetic selection

Genetic selection systems, such as the yeast two-hybrid system, are efficient methods to detect protein–protein and protein–ligand interactions. These systems have been further developed to assess negative interactions, such as inhibition, using the URA3 genetic selection marker. Previously, chemical complementation was used to assess positive selection in Saccharomyces cerevisiae. In this work, a new S. cerevisiae strain, called BAPJ69-4A, containing three selective markers ADE2, HIS3, and URA3 as well as the lacZ gene controlled by Gal4 response elements, was developed and characterized using the retinoid X receptor (RXR) and its ligand 9-cis retinoic acid (9cRA). Further characterization was performed using RXR variants and the synthetic ligand LG335. To assess the functionality of the strain, RXR was compared to the parent strain PJ69-4A in adenine, histidine, and uracil selective media. In positive selection, associating partners that lead to cell growth were observed in all media in the presence of ligand, whereas partners that did not associate due to the absence of ligand displayed no growth. Conversely, in negative selection, partners that did not associate in 5-FOA medium did not display cell death due to the lack of expression of the URA3 gene. The creation of the BAPJ69-4A yeast strain provides a high-throughput selection system, called negative chemical complementation, which can be used for both positive and negative selection, providing a fast, powerful tool for discovering novel ligand receptor pairs for applications in drug discovery and protein engineering.

Construction of covalent membrane protein complexes and high-throughput selection of membrane mimics.

The association of transmembrane (TM) helices underlies membrane protein structure and folding. Structural studies of TM complexes are limited by complex stability and the often time-consuming selection of suitable membrane mimics. Here, methodology for the efficient, preparative scale construction of covalent TM complexes and the concomitant high-throughput selection of membrane mimics is introduced. For the employed integrin αIIbβ3 model system, the methodology identified phospholipid bicelles, including their specific composition, as the best membrane mimic. The method facilitates structure determination by NMR spectroscopy as exemplified by the measurement of previously inaccessible residual dipolar couplings and (15)N relaxation parameters.

Stabilizing Proteins from Sequence Statistics: The Interplay of Conservation and Correlation in Triosephosphate Isomerase Stability

Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and high-throughput screens or selections are needed to stabilize proteins using directed evolution. Consensus mutations have proven effective for increasing stability, but these mutations are successful only about half the time. We set out to understand why some consensus mutations fail to stabilize, and what criteria might be useful to predict stabilization more accurately. Overall, consensus mutations at more conserved positions were more likely to be stabilizing in our model, triosephosphate isomerase (TIM) from Saccharomyces cerevisiae. However, positions coupled to other sites were more likely not to stabilize upon mutation. Destabilizing mutations could be removed both by removing sites with high statistical correlations to other positions and by removing nearly invariant positions at which “hidden correlations” can occur. Application of these rules resulted in identification of stabilizing mutations in 9 out of 10 positions, and amalgamation of all predicted stabilizing positions resulted in the most stable yeast TIM variant we produced (+8 °C). In contrast, a multimutant with 14 mutations each found to stabilize TIM independently was destabilized by 2 °C. Our results are a practical extension to the consensus concept of protein stabilization, and they further suggest the importance of positional independence in the mechanism of consensus stabilization.

New Technologies, Tools and Approaches for Improving Crop Breeding

New Technologies, Tools and Approaches for Improving Crop Breeding

A High-Throughput Screen for the Engineered Production of β-Lactam Antibiotics

High-throughput screens and selections have had profound impact on our ability to engineer proteins possessing new, desired properties. These methods are especially useful when applied to the modification of existing enzymes to create natural and unnatural products. In an advance upon existing methods we developed a high-throughput, genetically regulated screen for the in vivo production of β-lactam antibiotics using a green fluorescent protein (gfp) reporter. This assay proved reliable and sensitive and presents a dynamic range under which a wide array of β-lactam architectural subclasses can be detected. Moreover, the graded response elicited in this assay can be used to rank mutant activity. The utility of this development was demonstrated in vivo and then applied to the first experimental investigation of a putative catalytic residue in carbapenem synthase (CarC). Information gained about the mutability of this residue defines one parameter for enzymatic activity and sets boundaries for future mechanistic and engineering efforts.

Formulation Design and High-Throughput Excipient Selection Based on Structural Integrity and Conformational Stability of Dilute and Highly Concentrated IgG1 Monoclonal Antibody Solutions

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).