A phage display system designed to detect and study protein-protein interactions.

Analysing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described.     

Acidification-induced changes in Cx43 protein–protein interactions

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH-d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions

We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.     

Detection of a homotetrameric structure and protein–protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role in fungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was overexpressed in bacteria and a polyclonal antibody to this protein was produced. We identified a 180-kDa protein species reactive to the antibody produced in mice against the P. brasiliensis recombinant purified formamidase of 45 kDa. The 180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide mass fingerprinting using MS. The identical mass spectra generated by the 180 and the 45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formamidase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights into formamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism.     

Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.     

A helper phage to improve single-chain antibody presentation in phage display

We show here that the number of single-chain antibody fragments (scFv) presented on filamentous phage particles generated with antibody display phagemids can be increased by more than two orders of magnitude by using a newly developed helper phage (hyperphage). Hyperphage have a wild-type pIII phenotype and are therefore able to infect F(+) Escherichia coli cells with high efficiency; however, their lack of a functional pIII gene means that the phagemid-encoded pIII-antibody fusion is the sole source of pIII in phage assembly. This results in an considerable increase in the fraction of phage particles carrying an antibody fragment on their surface. Antigen-binding activity was increased about 400-fold by enforced oligovalent antibody display on every phage particle. When used for packaging a universal human scFv library, hyperphage improved the specific enrichment factor obtained when panning on tetanus toxin. After two panning rounds, more than 50% of the phage were found to bind to the antigen, compared to 3% when conventional M13KO7 helper phage was used. Thus, hyperphage is particularly useful in stoichiometric situations, when there is little chance that a single phage will locate the desired antigen.     

Analysis of PDZ domain-ligand interactions using carboxyl-terminal phage display

PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.     

A twin-arginine translocation (Tat)-mediated phage display system

The major limitation of conventional phage display is caused by its dependence on the Sec translocation pathway. All proteins displayed on filamentous phages must first be transported into the bacterial periplasm in an unfolded state via the Sec translocation machinery. Proteins that require a cytoplasmic environment and/or cytoplasmic components for folding, or that contain "stop transfer" signals, or reach their native state before they interact with the Sec proteins are not compatible with the Sec pathway. They can never be presented using conventional phage display. We have developed an alternative phage display system, termed the TPD system, which overcomes these limitations of conventional phage display by exploiting the properties of the twin-arginine translocation (Tat) pathway. The Tat pathway only exports folded proteins that have already attained their native conformation in the cytoplasm. We investigated the functional efficiency of the TPD system by displaying and panning for a mutant of the green fluorescent protein.     

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.     

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

The development of an electroporation based transfection method for Plasmodium falciparum has been very successful for the study of some genes but its efficiency remains very low. While alternative approaches have been documented, electroporation of infected red blood cells generally remains the method of choice for introducing DNA into P. falciparum. In this paper we compare four published transfection techniques in their ability to achieve stable transfections.     

Enhanced antibody affinity to Japanese encephalitis virus E protein by phage display

Obtaining antibodies with high affinity and specificity against antigens are required for the development of therapeutic and diagnostic antibodies. In this study, the contributions to binding affinity in the CDR2 and CDR3 regions of two monoclonal antibodies E3.3 and 2H2 were investigated by random mutagenesis in a phage-display synthetic oligonucleotide library. One high-affinity clone (CDR3-30) was obtained with a 3-fold increase of the dissociation constant, resulting from the changes in amino acids at residues 95, 97, and 98 in the CDRH3 region. Analysis of the predicted structure by modeling suggested that the contributions of mutated residues in the CDR3 region to the binding affinity involved not only complementarity between antigen and CDR3, but also interaction between heavy and light chains. The information gained from this study may benefit the design of vaccines and therapeutic antibodies against Japanese encephalitis virus infection.     

Selection for improved protein stability by phage display.

A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display.     

The yeast split-ubiquitin system to study chloroplast membrane protein interactions

Each photosynthetic complex within the thylakoid membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein–protein interactions of thylakoid membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the thylakoid membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF₀III, suggesting an important role of Alb3 in the assembly of photosynthetic thylakoid membrane complexes.     

Expanding the versatility of phage display I: efficient display of peptide-tags on protein VII of the filamentous phage

Background

Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display.

Methodology/Principal Findings

Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS₆ or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether.

Conclusions/Significance

Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations.     

Plasmodium-mosquito interactions,phage display libraries and transgenic mosquitoes impaired for malaria transmission

Malaria continues to kill millions of people every year and new strategies to combat this disease are urgently needed. Recent advances in the study of the mosquito vector and its interactions with the malaria parasite suggest that it may be possible to genetically manipulate the mosquito in order to reduce its vectorial capacity. Here we review the advances made to date in four areas: (1) the introduction of foreign genes into the mosquito germ line; (2) the characterization of tissue-specific promoters; (3) the identification of gene products that block development of the parasite in the mosquito; and (4) the generation of transgenic mosquitoes impaired for malaria transmission. While initial results show great promise, the problem of how to spread the blocking genes through wild mosquito populations remains to be solved.