Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries.

Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambda D-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).

ADAM-HCV, a new-concept diagnostic assay for antibodies to hepatitis C virus in serum.

We screened phage libraries using sera from noninfected individuals and patients infected by hepatitis C virus (HCV). By applying different selection and maturation strategies, we identified a wide collection of efficient phage-borne ligands for HCV-specific antibodies. The selected ligands retained their antigenic properties when expressed as multimeric synthetic peptides. Peptides that mimic several immunodominant epitopes of the virus were used to develop a novel type of diagnostic assay which efficiently detects antibodies to HCV in serum. This type of analysis provides a conclusive diagnosis for many patients identified as indeterminate according to presently available serological assays.

'In vitro evolution' of ligands for HCV-specific serum antibodies.

We developed a strategy to improve the properties of ligands selected from phage-displayed random peptide libraries. A site-directed mutagenesis protocol that introduces mutations and extends the size of a target sequence has been set up to generate diversity in a single or in a population of clones. The pool of mutants thus created is screened to identify variants with the desired properties. We refer to this strategy as in vitro evolution' of ligands. Here we report the application of this in vitro evolution protocol to the identification of improved ligands for HCV-specific serum antibodies. A single clone or population of clones were processed to generate a secondary library. Screening of these libraries with sera from HCV-infected patients identified peptides with an enhanced and broadened ability to detect HCV-specific serum antibodies.

The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity.

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.

Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module.

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.

Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage.

The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface.

The genes encoding the transcription factor yTAFII60, the G4p1 protein and a putative glucose transporter are contained in a 12.3 kb DNA fragment on the left arm of Saccharomyces cerevisiae chromosome VII.

We report the nucleotide sequence of a DNA fragment of 12,325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII. Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids. Five ORFs are significantly homologous to known proteins. The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAFII60. The G3075 ORF is 47.8% identical to the hypothetical yeast protein YB88. G3089 shows 36.7% identity to the eel calmodulin. G3085 shows 94.9% identity with the published sequence of the quadruplex DNA binding protein G4p1. G3090 reveals 46.7% identity with the probable glucose transport protein yBR1625.

[Detection of bovine infectious rhinotracheitis virus by hybridization using nonradioactive DNA-probes]. / Vyiavlenie virusa infektsionnogo rinotrakheita krupnogo rogatogo skota gibridizatsiei s neradioaktivnymi DNK-zondami.

Biotin-labeled DNA probes for infectious bovine rhinotracheitis virus also known as bovine herpesvirus-1 (BHV-1) have been developed. The procedure is based on dot-blot hybridization using biotin-labeled bacteriophage M13 and plasmid probes containing cloned PstI and EcoRI-PstI restriction fragments of viral genome. The probes obtained were used to detect viral nucleic acids in specimens of bovine spermatic fluid or nasal swabs of calves. The method is simple and rapid, taking less than 24 h, and is highly specific and sensitive, this recommending it for practical veterinary.

Modifying filamentous phage capsid: limits in the size of the major capsid protein.

Ff filamentous phages are long thin cylindrical structures that infect bacteria displaying the F pilus and replicate without lysing the host. These structures are exploited to display peptides by fusing them to the amino terminus of either the bacterial receptor protein (pIII) or the major coat protein (pVIII). We have analysed a vast collection of phage mutants containing substitutions and insertions in the amino terminus of pVIII to ask whether any chemical group of this solvent exposed region of the phage capsid has any key function in the phage life cycle. Any of the five amino-terminal residues can be substituted by most amino acids without affecting phage assembly suggesting that this region does not play any essential role in morphogenesis. However, a deletion of three residues delta (Gly3Asp4Asp5) results in a phage clone with an decreased ability to produce infective particles. By engineering phages designed to display peptides by fusion to the amino terminus of the major coat protein we have found that phage viability is affected by peptide length while peptide sequence plays a minor "tuning" role. Most peptides of six residues are tolerated irrespective of their sequence while only 40% of the phages carrying an amino-terminal extension of eight residues can form infective particles. This fraction drops to 20% and 1% when we attempt to insert peptides 10 and 16 amino acids long. We have used this information to build phage libraries where each phage displays approximately 2700 copies of a different octapeptide all over the phage surface.

[Analysis of peptide fragment insertions in the basic proteins of the bacteriophage M13, f1, and fd envelopes. Interconnection of structural characteristics of the envelope protein and viability of mutant phages]. / Analiz vstroek peptidnykh fragmentov v osnovnoi belok obolochki bakteriofagov M13, f1, i fd. Vzaimosviaz' strukturnykh kharakteristik belka obolochki i zhiznesposobnosti mutantnykh fagov.

An analysis of 12 peptide fragment insertions into the major coat protein (protein pVIII) of bacteriophages M13, f1 and fd has been done. To elucidate the relations between protein structural characteristics and viability of mutant phages, we used the program Pro-Anal. Correlations were found between phage viability and different physicochemical and structural characteristics of protein N-termini. Thus peptide insertions in nonviable phages have high indexes of alpha-helicity, volumes, and polarity as well as high moments (alpha-helical and beta-structural) of isoelectric point. On the other hand, high beta-turn indexes are correlated with viability. The most important factor which determines phage viability is the lack of positively charged amino acid residues on the C-terminal ends of peptide insertions. The correlations found hold for the pVIII proteins of four related phages-M13, IKe, If1 and I2-2. Based on these results, the rule of obtaining viable mutant phages with insertions in the major coat protein is suggested. A new site is described for peptide insertions--upstream of the first amino acid residue of the mature protein sequence.

Design of specific immunogens using filamentous phage as the carrier.

Earlier, we developed an expression vector allowing exposure of short peptides on the surface of bacteriophage M13. It was used to obtain a recombinant phage carrying an antigenic determinant of HIV1 p17 Gag protein. Immunoglobulin elicited by immunizing rabbits with the phage reacted with the 17-kDa core protein of the virus and with its polyprotein precursor, p55, on Western blots of HIV1 viral proteins. The results of present experiments may be useful in vaccine development.

[Preparation of a specific immunogen based on bacteriophage M13]. / Poluchenie spetsificheskogo immunogena na osnove bakteriofaga M13.

Earlier we developed an expression vector on the basis of bacteriophage M13 allowing the exposure of short peptides on the virion surface. It was used to obtain a recombinant phage carrying the antigenic determinant of HIVI gag protein p17. This phage was tested as immunogen in rabbits. It was shown by ELISA that Ig against the fusion phage reacted with the 17-kDa core protein of the virus and with its polyprotein precursor p55 on strips activated by the transfer of HIVI viral proteins. These data may be used in vaccine development.

Inserting foreign peptides into the major coat protein of bacteriophage M13.

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.

[Structure of virions of the M13 phage containing chimeric B-protein molecules]. / Izuchenie struktury virionov faga M13, soderzhashchikh molekuly khimernykh B-belkov.

We have studied the virion structure of M13 strains (M13B1, M13BOM1, M13BOM2, M13BOL1) with chimeric variants of B-protein. Data concerning the spatial structure of chimeric B-protein molecules and their interaction with intraphage DNA were obtained. The phage contour lengths were measured under electron microscope and the DNA/protein ratios were obtained by spectrophotometry. These data testified that the insertion of foreign peptide affected neither DNA packaging nor the compactness of molecular arrangement of proteins in the virion. By linear dichroism and fluorescence spectra of phages it was determined, that the insert can influence the polarity of amino acid environment and the orientation of amino acids in the B-protein central part. It was shown by quenching of phage fluorescence by KI that the inward or outward amino acids location in the capsid is invariable. The carboxyl residues have been titrated in the phage strains by Auramine O. It was shown that there is no correlation between the number of the titrated carboxyl groups and the number of the carboxyl groups as a whole.

[The use of filamentous phage M13 in protein engineering]. / Ispol'zovanie nitchatogo faga M13 dlia belkovoi inzhenerii.

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.