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1.
Int J Parasitol ; 31(14): 1659-68, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11730793

ABSTRACT

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).


Subject(s)
Antigens, Protozoan/immunology , Epitopes, B-Lymphocyte/immunology , Immunodominant Epitopes/immunology , Toxoplasma/immunology , Toxoplasmosis/immunology , Amino Acid Sequence , Animals , Antigens, Protozoan/genetics , Bacteriophage lambda/chemistry , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Enzyme-Linked Immunosorbent Assay , Epitopes, B-Lymphocyte/genetics , Female , Gene Library , Glutathione Transferase/metabolism , Humans , Immunodominant Epitopes/genetics , Molecular Sequence Data , Peptide Library , Polymerase Chain Reaction , Pregnancy , Sequence Homology, Amino Acid , Toxoplasma/chemistry , Toxoplasma/genetics
2.
Eur J Biochem ; 268(17): 4758-68, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11532012

ABSTRACT

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.


Subject(s)
Hepacivirus/immunology , Hepatitis C Antibodies/blood , Amino Acid Sequence , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoblotting , Ligands , Molecular Mimicry/immunology , Molecular Sequence Data , Peptide Library , Sequence Homology, Amino Acid
4.
Biol Chem ; 381(3): 245-54, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10782995

ABSTRACT

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.


Subject(s)
Antibodies, Viral/metabolism , Hepacivirus/immunology , Peptide Library , Antibodies, Viral/genetics , Cloning, Molecular , Directed Molecular Evolution , Enzyme-Linked Immunosorbent Assay , Humans , Ligands , Mutagenesis, Site-Directed
5.
J Biol Chem ; 274(45): 32001-7, 1999 Nov 05.
Article in English | MEDLINE | ID: mdl-10542231

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Nerve Tissue Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Proline/metabolism , src Homology Domains , Amino Acid Sequence , Binding Sites , Binding, Competitive , Enzyme-Linked Immunosorbent Assay , Humans , Molecular Sequence Data , Peptide Library
6.
Genes Dev ; 11(17): 2239-49, 1997 Sep 01.
Article in English | MEDLINE | ID: mdl-9303539

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Vesicular Transport , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Nuclear Pore Complex Proteins , Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA-Binding Proteins , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Binding Sites/genetics , Calcium-Binding Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/genetics , Drosophila Proteins , Gene Products, rex/chemistry , Gene Products, rex/genetics , Gene Products, rex/metabolism , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , Juvenile Hormones/chemistry , Juvenile Hormones/genetics , Juvenile Hormones/metabolism , Molecular Sequence Data , Open Reading Frames , Phosphoproteins/genetics , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction
7.
Biol Chem ; 378(6): 517-21, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9224932

ABSTRACT

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.


Subject(s)
Capsid/chemistry , Capsid/genetics , Inoviridae/chemistry , Inoviridae/genetics , Peptide Library , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Base Sequence , Binding Sites, Antibody , Capsid/metabolism , Cloning, Molecular , Directed Molecular Evolution , Inoviridae/metabolism , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/chemical synthesis , Triazines/metabolism
8.
Yeast ; 13(1): 85-91, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9046090

ABSTRACT

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.


Subject(s)
Chromosomes, Fungal/genetics , DNA-Binding Proteins/genetics , Genes, Fungal/genetics , Monosaccharide Transport Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , TATA-Binding Protein Associated Factors , Transcription Factor TFIID , Transcription Factors/genetics , Amino Acid Sequence , Calmodulin , Chromosome Mapping , Genome, Fungal , Molecular Sequence Data , Open Reading Frames , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid
9.
Vopr Virusol ; 40(6): 279-82, 1995.
Article in Russian | MEDLINE | ID: mdl-8686268

ABSTRACT

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.


Subject(s)
DNA Probes , Herpesvirus 1, Bovine/isolation & purification , Nucleic Acid Hybridization , Animals , Bacteriophage M13/genetics , Cattle , Cell Line , Genome, Viral , Herpesvirus 1, Bovine/genetics , Infectious Bovine Rhinotracheitis/diagnosis , Plasmids , Sensitivity and Specificity
10.
J Mol Biol ; 248(4): 835-44, 1995 May 12.
Article in English | MEDLINE | ID: mdl-7752244

ABSTRACT

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.


Subject(s)
Capsid/genetics , Genetic Vectors/genetics , Inovirus/genetics , Amino Acid Sequence , Base Sequence , Capsid/chemistry , Genes, Viral/genetics , Inovirus/growth & development , Molecular Sequence Data , Oligodeoxyribonucleotides/genetics , Recombinant Fusion Proteins , Sequence Deletion/physiology , Viral Structural Proteins/genetics
11.
Mol Biol (Mosk) ; 27(6): 1345-55, 1993.
Article in Russian | MEDLINE | ID: mdl-8283982

ABSTRACT

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.


Subject(s)
Bacteriophage M13/chemistry , Bacteriophages/chemistry , Capsid/chemistry , Mutation , Peptide Fragments/chemistry , Amino Acid Sequence , Bacteriophage M13/genetics , Bacteriophages/genetics , Molecular Sequence Data , Protein Conformation
12.
Gene ; 128(1): 85-8, 1993 Jun 15.
Article in English | MEDLINE | ID: mdl-7685305

ABSTRACT

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.


Subject(s)
Bacteriophage M13/genetics , Gene Products, gag/biosynthesis , Gene Products, gag/immunology , HIV-1/genetics , Amino Acid Sequence , Ampicillin Resistance/genetics , Animals , Base Sequence , Blotting, Western , Capsid/biosynthesis , Capsid/immunology , Cloning, Molecular/methods , Epitopes/analysis , Epitopes/biosynthesis , Epitopes/genetics , Escherichia coli/genetics , Gene Products, gag/genetics , Genetic Vectors , Molecular Sequence Data , Rabbits/immunology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunology , Recombination, Genetic
13.
Mol Biol (Mosk) ; 27(3): 561-8, 1993.
Article in Russian | MEDLINE | ID: mdl-7686250

ABSTRACT

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.


Subject(s)
AIDS Vaccines , Bacteriophage M13/genetics , Epitopes/immunology , Gene Products, gag/immunology , Genetic Vectors , HIV Antigens/immunology , Vaccines, Synthetic/genetics , Viral Proteins , Amino Acid Sequence , Animals , Base Sequence , DNA, Single-Stranded , Enzyme-Linked Immunosorbent Assay , Gene Products, gag/genetics , HIV Antigens/genetics , HIV-1/immunology , Molecular Sequence Data , Rabbits , Vaccines, Synthetic/immunology , gag Gene Products, Human Immunodeficiency Virus
14.
FEBS Lett ; 301(3): 322-4, 1992 Apr 27.
Article in English | MEDLINE | ID: mdl-1577174

ABSTRACT

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.


Subject(s)
Bacteriophages/genetics , Capsid/genetics , Mutagenesis, Site-Directed , Peptides/genetics , Amino Acid Sequence , Bacteriophages/pathogenicity , Base Sequence , Molecular Sequence Data , Plasmids
15.
Mol Biol (Mosk) ; 25(6): 1497-503, 1991.
Article in Russian | MEDLINE | ID: mdl-1813797

ABSTRACT

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.


Subject(s)
Bacteriophages , Viral Fusion Proteins/metabolism , Virion , Amino Acid Sequence , Bacteriophages/metabolism , Bacteriophages/ultrastructure , Chimera , DNA, Viral/metabolism , Microscopy, Electron , Molecular Sequence Data , Protein Conformation , Spectrum Analysis , Viral Fusion Proteins/genetics , Virion/metabolism , Virion/ultrastructure
16.
Mol Biol (Mosk) ; 24(2): 530-5, 1990.
Article in Russian | MEDLINE | ID: mdl-2362594

ABSTRACT

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.


Subject(s)
Capsid Proteins , Capsid/genetics , Coliphages/genetics , DNA, Viral/genetics , Membrane Proteins/genetics , Protein Engineering , Amino Acid Sequence , Base Sequence , Blotting, Southern , Molecular Sequence Data , Mutation , Recombination, Genetic
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