Fluorescent probe for high‐throughput screening of membrane protein expression

Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence‐detection size‐exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false‐positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine‐tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G‐protein coupled adenosine A2a receptor were readily identified from crude detergent‐extracts of a library of construct variants transiently produced in suspension‐adapted HEK293‐6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture.     

Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins

Formation of well-ordered crystals of membrane proteins is a bottleneck for structure determination by X-ray crystallography. Nevertheless, one can increase the probability of successful crystallization by precrystallization screening, a process by which one analyzes the monodispersity and stability of the protein-detergent complex. Traditionally, this has required microgram to milligram quantities of purified protein and a concomitant investment of time and resources. Here, we describe a rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass. We show the application of this precrystallization screening to four membrane proteins derived from prokaryotic or eukaryotic organisms.     

G‐protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H1‐histamine or M2‐muscarinic receptors

This paper describes a novel strategy to create a microarray of G‐protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁‐histamine receptor and the M₂‐muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol‐modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM‐D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self‐sorting liposome array of GPCRs which would underpin a variety of future novel applications.     

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF‐hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF‐hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N‐terminal myristoyl group may be important for membrane binding. Non‐conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.     

Genetic selection system for improving recombinant membrane protein expression in E. coli

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C‐terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I‐CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75‐fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90‐fold.     

Cystine‐knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4)

Cystine‐knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine‐knot peptide MCoTI‐II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine‐knot trypsin inhibitor into a CTLA‐4 binder by screening a library of variants using yeast surface display. A set of cystine‐knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400‐fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Cell‐free expression profiling of E. coli inner membrane proteins

The high versatility and open nature of cell‐free expression systems offers unique options to modify expression environments. In particular for membrane proteins, the choice of co‐translational versus post‐translational solubilization approaches could significantly modulate expression efficiencies and even sample qualities. The production of a selection of 134 α‐helical integral membrane proteins of the Escherichia coli inner membrane proteome focussing on larger transporters has therefore been evaluated by a set of individual cell‐free expression reactions. The production profiles of the targets in different cell‐free expression modes were analyzed independently by three screening strategies. Translational green fluorescent protein fusions were analyzed as monitor for the formation of proteomicelles after cell‐free expression of membrane proteins in the presence of detergents. In addition, two different reaction configurations were implemented and performed either by robotic semi‐throughput approaches or by individually designed strategies. The expression profiles were specified for the particular cell‐free modes and overall, the production of 87% of the target list could be verified and approximately 50% could already be synthesized in preparative scales. The expression of several selected targets was up‐scaled to milliliter volumes and milligram amounts of production. As an example, the flavocytochrome YedZ was purified and its sample quality was demonstrated.     

A rapid expression and purification condition screening protocol for membrane protein structural biology

Membrane proteins control a large number of vital biological processes and are often medically important—not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high‐resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time‐consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96‐well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large‐scale production of detergent‐solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large‐scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.     

Rapid,robotic, small‐scale protein production for NMR screening and structure determination

Three‐dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time‐consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi‐automated protocol for isotopically‐labeled protein production using the Maxwell‐16, a commercially available bench top robot, that allows for single‐step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different ¹⁵N‐labeled proteins, accelerating the validation process by more than 10‐fold. The yield from a single channel of the Maxwell‐16 is sufficient for acquisition of a high‐quality 2D ¹H‐¹⁵N‐HSQC spectrum using a 3‐mm sample cell and 5‐mm cryogenic NMR probe. Maxwell‐16 screening of a control group of proteins reproduced previous validation results from conventional small‐scale expression screening and large‐scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par‐3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U‐¹⁵N,¹³C] protein prepared using the Maxwell‐16. This novel semi‐automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale‐up steps.     

Inhibition of Orientia tsutsugamushi infection by a truncated recombinant 56‐kDa outer membrane protein

Aims: The objective of this study was to evaluate recombinant 56‐kDa outer membrane protein as a potential inhibitor to infection from Orientia tsutsugamushi. Methods and Results: The 56‐kDa protein was cloned and expressed in an Escherichia coli system, and the degree of target cell attachment to immobilized 56‐kDa protein was measured in a cell adhesion assay. The results showed that the 56‐kDa protein has an ability to attach HeLa cells. Furthermore, treatment of target cells with a truncated 56‐kDa 1–418 (amino acid residues) protein inhibited target cell infection by O. tsutsugamushi, demonstrated with an indirect immunofluorescence antibody assay. Conclusions: The truncated 56‐kDa protein (a.a. 1–418) plays an important role in O. tsutsugamushi infection, and the 56‐kDa protein could be useful and effective in the inhibition of O. tsutsugamushi attachment and infection. Significance and Impact of the Study: The attachment of the 56‐kDa protein to target cells was directly determined by in vitro adherence test, and the invasion of target cells by O. tsutsugamushi was inhibited by treating the target cells with a truncated 56‐kDa protein.     

Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris

The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate‐screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N‐methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing “jackpot” clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in‐culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.     

A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening

Optimization of membrane protein stability under different solution conditions is essential for obtaining crystals that diffract to high resolution. Traditional methods that evaluate protein stability require large amounts of material and are, therefore, ill suited for medium- to high-throughput screening of membrane proteins. Here we present a rapid and efficient fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS). In this method, the target protein is fused to GFP. Heated protein samples, treated with a panel of additives, are then analyzed by FSEC. FSEC-TS allows one to evaluate the thermostability of nanogram-to-microgram amounts of the target protein under?a variety of conditions without purification. We applied this method to the Danio rerio P2X4 receptor and Caenorhabditis elegans GluCl to screen ligands, ions, and lipids, including newly designed cholesterol derivatives. In the case of GluCl, the screening results were used to obtain crystals of the receptor in the presence of lipids.     

A structure-based strategy for discovery of small ligands binding to functionally unknown proteins: combination of in silico screening and surface plasmon resonance measurements

In the postgenomic era, many researchers and organizations have been engaged in structural and functional analyses of proteins. As a part of these efforts, searching for small organic compounds that bind specifically to target proteins is quite important. In this study, we have developed a rational strategy for ligand discovery based on the three-dimensional structures of target proteins, which were elucidated by X-ray crystallography and nuclear magnetic resonance spectroscopy. The strategy has three features: (i) rapid selection of candidate compounds by in silico screening, (ii) automated preparation of sample solutions with robotics, and (iii) reliable evaluation of the candidates with surface plasmon resonance. Applying the strategy to a protein, At2g24940 from Arabidopsis thaliana, we discovered four small ligands out of a commercially available library of about 150 000 compounds. Although these compounds had only weak affinities to the target protein, with dissociation constants ranging from 68 to 120 microM, they apparently possess common structural features. They would be leads for the development of specific inhibitors/drugs for At2g24940, and provide important clues toward elucidation of the protein function.     

Tandem Cell‐Free Protein Synthesis as a Tool for Rapid Screening of Optimal Molecular Chaperones

A simple and flexible method is developed for rapid screening of molecular chaperones that enhance the functional expression of recombinant proteins. A panel of molecular chaperones are transiently expressed in a reaction mixture of cell‐free protein synthesis and then a target protein is subsequently expressed in the presence of these presynthesized molecular chaperones. The biological activity of the cell‐free synthesized target protein is compared to identify the effective molecular chaperones. This strategy successfully identifies individual and combinations of bacterial molecular chaperones that markedly improved the functional expression of horseradish peroxidase. The authors believe that the presented strategy provides a versatile platform for the optimal production of functional proteins, and can also be extended to studies of other interacting proteins.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

Fast identification of thermostable beta‐glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach

Engineering costly cellulases on natural cellulosic substrates is of importance for emerging biomass‐based biorefineries. Directed enzyme evolution is becoming a popular tool, but identification of desired mutants from a large mutant library remains challenging sometimes. In this work, we demonstrated a novel combinatorial selection/screening strategy for finding thermostable beta‐glucosidase on its natural substrate—cellobiose. First, selection was conducted through complementation of beta‐glucosidase for non‐cellobiose‐utilizing Escherichia coli so that only the cells expressing active beta‐glucosidase can grow on a M9 synthetic medium with cellobiose as the sole carbon source (selection plate). Second, the clones on the selection plates were duplicated by using nylon membranes. After heat treatment, the nylon membranes were overlaid on M9/cellobiose screening plates so that remaining activities of thermostable beta‐glucosidase mutants hydrolyzed cellobiose on the screening plates to glucose. Third, the growth of an indicator E. coli strain that can utilize glucose but not cellobiose on the screening plates helped detect the thermostable beta‐glucosidase mutants on the selection plates. Several thermostable mutants were identified from a random mutant library of the Paenibacillus polymyxa beta‐glucosidase. The most thermostable mutant A17S had an 11‐fold increase in the half‐life of thermoinactivation at 50°C. Biotechnol. Bioeng. 2009;103: 1087–1094. © 2009 Wiley Periodicals, Inc.     

Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans

Abstract

Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.     

Dissecting the Photoprotective Mechanism Encoded by the flv4‐2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

In Synechocystis sp. PCC 6803, the flv4‐2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4‐2 operon mutants. In the ?sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ?sll0218‐flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ?sll0218 and ?sll0218‐flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218‐lacking mutants. The YFP‐tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA‐defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ?flv4‐2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.     

Uncoupling of bait‐protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP‐1 and the PKA Cβ1 subunit

An expression‐uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP‐bait protein from the target cells. Here, the TAP‐tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue‐specific binding partners of the protein kinase A catalytic subunit Cβ1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor‐dependent interaction of Cβ1 with the cell cycle and apoptosis regulatory protein‐1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell‐ and tissue‐specific protein complexes.