Automated ELISA for potency measurements of therapeutic antibodies and antibody fragments.

Potency assays are essential for the development and quality control of biopharmaceutical drugs, but they are often a time limiting factor due to manual handling steps and consequently low analytical throughput. On the other hand, automation of potency assays can be challenging due to their complexity and the use of biological materials. ELISA (enzyme-linked immunosorbent assay) is widely used for potency determination and is a good candidate for automation as all ELISA types depend on the same basic steps: coating, blocking, sample incubation, detection, and signal measurement. Nevertheless, ELISA for relative potency measurements still require drug-specific development and assay validation thereby complicating automation efforts. To simplify potency testing by ELISA, we first developed a manual protocol generally applicable to different drugs and then adapted this protocol for automated measurements. We identified unexpected critical parameters which had to be adapted to transfer the manual ELISA to an automated liquid handling system and we demonstrated that gravimetric sample dilution is unnecessary with the automated protocol. Both manual and automated protocols were validated and compared using multiple biotherapeutics. The automated protocol showed similar or higher precision and accuracy when compared to the manual method.

Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins.

Real-time quantitative PCR (qPCR) is important for quantification of residual host cell DNA (resDNA) in therapeutic protein preparations. Typical qPCR protocols involve DNA extraction steps complicating sample handling. Here, we describe a "direct qPCR" approach without DNA extraction. To avoid interferences of DNA polymerase with a therapeutic protein, proteins in the samples were digested with proteinase K (PK) in the presence of sodium dodecyl sulfate (SDS). Tween 20 and NaCl were included to minimize precipitation of therapeutic proteins in the PK/SDS mix. After PK treatment, the solution was applied directly for qPCR. Inhibition of DNA polymerase by SDS was prevented by adding 2% (v/v) of Tween 20 to the final qPCR mix. The direct qPCR approach was evaluated for quantification of resDNA in therapeutic proteins manufactured in Chinese hamster ovary (CHO) host cells. First, direct qPCR was compared with qPCR applied on purified DNA ("extraction qPCR"). For both qPCRs, the same CHO-specific primers and probes were used. Comparable residual DNA levels were detected with both PCR approaches in purified and highly concentrated drug proteins as well as in in-process-control samples. Finally, the CHO-specific direct qPCR protocol was validated according to ICH guidelines and applied for 25 different therapeutic proteins. The specific limits of quantification were 0.1-0.8ppb for 24 proteins, and 2.0ppb for one protein. General applicability of the direct qPCR was demonstrated by applying the sample preparation protocol for quantification of resDNA in therapeutic proteins manufactured in other hosts such as Escherichia coli and mouse cells.

Uncovering Arabidopsis membrane protein interactome enriched in transporters using mating-based split ubiquitin assays and classification models.

High-throughput data are a double-edged sword; for the benefit of large amount of data, there is an associated cost of noise. To increase reliability and scalability of high-throughput protein interaction data generation, we tested the efficacy of classification to enrich potential protein-protein interactions. We applied this method to identify interactions among Arabidopsis membrane proteins enriched in transporters. We validated our method with multiple retests. Classification improved the quality of the ensuing interaction network and was effective in reducing the search space and increasing true positive rate. The final network of 541 interactions among 239 proteins (of which 179 are transporters) is the first protein interaction network enriched in membrane transporters reported for any organism. This network has similar topological attributes to other published protein interaction networks. It also extends and fills gaps in currently available biological networks in plants and allows building a number of hypotheses about processes and mechanisms involving signal-transduction and transport systems.

Development of an assay for Complex I/Complex III of the respiratory chain using solid supported membranes and its application in mitochondrial toxicity screening in drug discovery.

Membrane-bound transporter proteins are involved in cell signal transduction and metabolism as well as influencing key pharmacological properties such as drug bioavailability. The functional activity of transporters that belong to the group of electrically active membrane proteins can be directly monitored using the solid-supported membrane-based SURFE(2)R™ technology (SURFace Electrogenic Event Reader; Scientific Devices Heidelberg GmbH, Heidelberg, Germany). The method makes use of membrane fragments or vesicles containing transport proteins adsorbed onto solid-supported membrane-covered electrodes and allows the direct measurement of their activity. This technology has been used to develop a robust screening compatible assay for Complex I/Complex III, key components of the respiratory chain in 96-well microtiter plates. The assay was screened against 1,000 compounds from the ComGenex Lead-like small molecule library to ascertain whether mitochondrial liabilities might be an underlying, although undesirable feature of typical commercial screening libraries. Some 105 hits (compounds exhibiting >50% inhibition of Complex I/Complex III activity at 10 µM) were identified and their activities were subsequently confirmed in duplicate, yielding a confirmation rate of 68%. Analysis of the confirmed hits also provided evidence of structure-activity relationships and two compounds from one structural class were further evaluated in dose-response experiments. This study provides evidence that profiling of compounds for potential mitochondrial liabilities, even at an early stage of drug discovery, may be a necessary additional quality filter that should be considered during the compound screening and profiling cascade.

N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1.

AMMONIUM TRANSPORTER (AMT) proteins are conserved in all domains of life and mediate the transport of ammonium or ammonia across cell membranes. AMTs form trimers and use intermolecular interaction between subunits to regulate activity. So far, binding forces that stabilize AMT protein complexes are not well characterized. High temperature or reducing agents released mono- and dimeric forms from trimeric complexes formed by AMT1;1 from Arabidopsis and tomato. However, in the paralogue LeAMT1;3, trimeric complexes were not detected. LeAMT1;3 differs from the other AMTs by an unusually short N-terminus, suggesting a role for the N-terminus in oligomer stability. Truncation of the N-terminus in LeAMT1;1 destabilized the trimer and led to loss of functionality when expressed in yeast. Swapping of the N-terminus between LeAMT1;1 and LeAMT1;3 showed that sequences in the N-terminus of LeAMT1;1 are necessary and sufficient for stabilization of the interaction among the subunits. Two N-terminal cysteine residues are highly conserved among AMT1 transporters in plants but are lacking in LeAMT1;3. C3S or C27S variants of LeAMT1;1 showed reduced complex stability, which coincided with lower transport capacity for the substrate analogue methylammonium. Both cysteine-substituted LeAMT1;1 variants showed weaker interactions with the wildtype as determined by a quantitative analysis of the complex stability using the mating-based split-ubiquitin assay. These data indicate that the binding affinity of AMT1 subunits is stabilized by cysteines in the N-terminus and suggest a role for disulphide bridge formation via apoplastic N-terminal cysteine residues.

Electrophysiology of respiratory chain complexes and the ADP-ATP exchanger in native mitochondrial membranes.

Transport of protons and solutes across mitochondrial membranes is essential for many physiological processes. However, neither the proton-pumping respiratory chain complexes nor the mitochondrial secondary active solute transport proteins have been characterized electrophysiologically in their native environment. In this study, solid-supported membrane (SSM) technology was applied for electrical measurements of respiratory chain complexes CI, CII, CIII, and CIV, the F(O)F(1)-ATPase/synthase (CV), and the adenine nucleotide translocase (ANT) in inner membranes of pig heart mitochondria. Specific substrates and inhibitors were used to validate the different assays, and the corresponding K(0.5) and IC(50) values were in good agreement with previously published results obtained with other methods. In combined measurements of CI-CV, it was possible to detect oxidative phosphorylation (OXPHOS), to measure differential effects of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the respective protein activities, and to determine the corresponding IC(50) values. Moreover, the measurements revealed a tight functional coupling of CI and CIII. Coenzyme Q (CoQ) analogues decylubiquinone (DBQ) and idebenone (Ide) stimulated the CII- and CIII-specific electrical currents but had inverse effects on CI-CIII activity. In summary, the results describe the electrophysiological and pharmacological properties of respiratory chain complexes, OXPHOS, and ANT in native mitochondrial membranes and demonstrate that SSM-based electrophysiology provides new insights into a complex molecular mechanism of the respiratory chain and the associated transport proteins. Besides, the SSM-based approach is suited for highly sensitive and specific testing of diverse respiratory chain modulators such as inhibitors, CoQ analogues, and uncoupling agents.

Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes.

Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes.

Solid-supported membrane technology for the investigation of the influenza A virus M2 channel activity.

Influenza A virus encodes an integral membrane protein, A/M2, that forms a pH-gated proton channel that is essential for viral replication. The A/M2 channel is a target for the anti-influenza drug amantadine, although the effectiveness of this drug has been diminished by the appearance of naturally occurring point mutations in the channel pore. Thus, there is a great need to discover novel anti-influenza therapeutics, and, since the A/M2 channel is a proven target, approaches are needed to screen for new classes of inhibitors for the A/M2 channel. Prior in-depth studies of the activity and drug sensitivity of A/M2 channels have employed labor-intensive electrophysiology techniques. In this study, we tested the validity of electrophysiological measurements with solid-supported membranes (SSM) as a less labor-intensive alternative technique for the investigation of A/M2 ion channel properties and for drug screening. By comparing the SSM-based measurements of the activity and drug sensitivity of A/M2 wild-type and mutant channels with measurements made with conventional electrophysiology methods, we show that SSM-based electrophysiology is an efficient and reliable tool for functional studies of the A/M2 channel protein and for screening compounds for inhibitory activity against the channel.

The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS).

Dynamic and reversible protein-protein interactions have a pivotal function in all living cells. For instance, protein-protein interactions are involved in the assembly and regulation of multimeric enzymes and transcription factors, various signal response pathways, intracellular sorting and movement of proteins and membrane vesicles, cell-to-cell protein transport, and many others. Here we provide a detailed protocol for the mating-based split-ubiquitin system (mbSUS), which is a sensitive and user-friendly alternative to the classical yeast two-hybrid system in particular. mbSUS relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions. Thus, mbSUS is predominantly suitable for the determination of full-length proteins localized in the cytoplasm and in or at membrane compartments, without the need for their truncation and nuclear mislocation. In addition, we present a set of Gateway compatible mbSUS vectors that allow the rapid generation of constructs for fast and efficient interaction studies. An additional vector is introduced that allows the extension of mbSUS for the analysis of oligomeric protein complex formation and competition assays in vivo. In summary, mbSUS provides an additional versatile tool for protein-protein interaction studies, which is complementary to in planta assays such as BiFC and FRET.

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli.

The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H(+)-dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.

Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene receptor family members.

The gaseous phytohormone ethylene regulates many developmental processes and responses to environmental conditions in higher plants. In Arabidopsis thaliana, ethylene perception and initiation of signaling are mediated by a family of five receptors which are related to prokaryotic two-component sensor histidine kinases. The transient expression of fluorescence-tagged receptors in tobacco (Nicotiana benthamiana) epidermal leaf cells demonstrated that all ethylene receptors are targeted to the ER endomembrane network and do not localize to the plasmalemma. In support of in planta overlay studies, the ethylene receptors form homomeric and heteromeric protein complexes at the ER in living plant cells, as shown by membrane recruitment assays. A comparable in vivo interaction pattern was found in the yeast mating-based split-ubiquitin system. The overlapping but distinct expression pattern of the ethylene receptor genes suggests a differential composition of the ethylene receptor complexes in different plant tissues. Our findings may have crucial functional implications on the ethylene receptor-mediated efficiency of hormone perception, induction of signaling, signal attenuation and output.

Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1.

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.

Split-ubiquitin system for identifying protein-protein interactions in membrane and full-length proteins.

Protein-protein interactions play a fundamental role in the regulation of almost all cellular processes. Thus, the identification of interacting proteins can help to elucidate their function. The mating-based split-ubiquitin system (mbSUS) uses yeast as a test organism to identify potential interactions between full-length membrane proteins or between a full-length membrane protein and a soluble protein. The mbSUS can also be used to provide further evidence for protein-protein interactions detected with other methods and to map the interaction domains of selected proteins. The mbSUS is optimized for systematic screening approaches employing a mating-based approach, as typically used to determine protein interactions on a genomic scale. Construction of bait and prey fusions is simplified by adapting two different cloning procedures: (i) in vivo cloning in yeast, and (ii) Gateway cloning in E. coli. Protocols for small-scale interaction tests, as well as systematic approaches using sorted bait and prey arrays, are described.

Transporter assays using solid supported membranes: a novel screening platform for drug discovery.

Transporters are important targets in drug discovery. However, high throughput-capable assays for this class of membrane proteins are still missing. Here we present a novel drug discovery platform technology based on solid supported membranes. The functional principles of the technology are described, and a sample selection of transporter assays is discussed: the H(+)-dependent peptide transporter PepT1, the gastric proton pump, and the Na(+)/Ca(2+) exchanger. This technology promises to have an important impact on the drug discovery process.

K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions.

Organization of proteins into complexes is crucial for many cellular functions. However, most proteomic approaches primarily detect protein interactions for soluble proteins but are less suitable for membrane-associated complexes. Here we describe a mating-based split ubiquitin system (mbSUS) for systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins. mbSUS allows in vivo cloning of PCR products into a vector set, detection of interactions via mating, regulated expression of baits, and improved selection of interacting proteins. Cloning is simplified by introduction of lambda attachment sites for GATEWAY. Homo- and heteromeric interactions between Arabidopsis K(+) channels KAT1, AKT1, and AKT2 were identified. Tests with deletion mutants demonstrate that the C terminus of KAT1 and AKT1 is necessary for physical assembly of complexes. Screening of a sorted collection of 84 plant proteins with K(+) channels as bait revealed differences in oligomerization between KAT1, AKT1, and AtKC1, and allowed detection of putative interacting partners of KAT1 and AtKC1. These results show that mbSUS is suited for systematic analysis of membrane protein interactions.

Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters.

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.