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At least 19 recordsLinked to original sources
PubMed1989

M13 Bioprints: non-isotopic detection of individual-specific human DNA fingerprints with biotinylated M13 bacteriophage.

M13 bacteriophage was labelled by primer extension with biotin-11-dUTP and was successfully used as probe to study human hypervariable minisatellites (DNA fingerprints) in Southern blots. The method, which we called M13 Bioprints, presents two advantages over conventional DNA fingerprinting. First, it is easily accessible, since it utilizes the widely available M13 phage as a probe. Second, it makes use of biotin, which is safer, simpler and more economical than isotopic labels.

Bacteriophages↗
PubMed1975

Identification and characterization of the in vitro synthesized gene products of bacteriophage M13.

Bacteriophage M13 replicative form (RF) DNA was used to direct coupled transcription and translation in cell-free extracts prepared from Escherichia coli. By using RF DNA, isolated from cells infected with appropriate amber mutants of this phage, it has been possible to identify the products of genes I through IV. By using the same methods no gene-product relationship could be demonstrated for genes VI and VII. Coupled in vitro protein synthesis studies on RF-III DNA, a linear double-stranded DNA molecule, obtained after cleavage of either RF-I or RF-II DNA with the restriction endonuclease R.Hin11 from Haemophilus influenzae, indicated that the cleavage site for this enzyme is located in gene II. The in vitro products of both gene III and gene VIII are about 30 and six amino acids longer, respectively, than their native counterparts present within the virion. These results suggest that the latter proteins arise in vivo by cleavage of precursor molecules. Coupled transcription and translation studies on a DNA fragment which only contained the genetic information coding for gene IV protein, obtained after cleavage of RF DNA with the restriction endonuclease R.Hap11 from Haemophilus aphirophilus, indicated that a large number of the in vitro synthesized polypeptides are the result of premature chain termination.

Autoradiography↗
PubMed2004

Purification of filamentous bacteriophage M13 by expanded bed anion exchange chromatography.

In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho = 15 cm) of STREAMLINE DEAE (rho = 1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.

Adsorption↗
PubMed1994

Construction and characterization of M13 bacteriophages displaying gp120 binding domains of human CD4.

The envelope glycoprotein, gp120, on the surface of HIV interacts with the human CD4 molecule and thus helps the virus in gaining entry into the T-helper cells. To display the gp120 binding domains of human CD4 on the surface of the bacteriophage M13, two types of vectors have been constructed. In these, the first 176 amino acids of the human CD4 have been fused with the minor coat protein, gIIIp, of M13 bacteriophage for surface display. The Western blot analysis revealed that using the phage based vector, M13CD41923, all the copies of gIIIP (3-5 per virion) were present as fusion protein indicating multivalent display. In the phagemid based vector, phage particles were produced only upon infection of the cells carrying pVCCD43426, with the helper phage, M13KO7. Thus these phage particles carried both, the fusion protein as well as the unfused gIIIp, as shown by Western blot analysis. The presence of large amount of unfused gIIIp ensured that the phage particles did not display more than one fusion protein per phage particle, thus leading to monovalent display. Phage particles produced by both vectors could be captured on immobilized gp120, thereby showing that the displayed CD4 domains were functional.

Bacteriophage M13↗
PubMed1996

Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry.

The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments. Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides corresponding to the M13 bacteriophage sequence. More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported. In the present study, these feasibility demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing. The results show sequence determination for extension products up to 35 bases in length. Different desalting and purification procedures were investigated and it was found that salt could be efficiently reduced by removal of the template in a post-reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence.

Bacteriophage M13↗
PubMed1992

Gene V protein-mediated translational regulation of the synthesis of gene II protein of the filamentous bacteriophage M13: a dispensable function of the filamentous-phage genome.

Introduction of a deletion in the genome of wild-type M13 bacteriophage that eliminates translational repression of M13 gene II by its cognate gene V protein had no effect on phage viability. Furthermore, it was noted that gene V protein of phage IKe, a distant relative of M13, does not function as a translational repressor of its cognate gene II protein. The data strongly indicate that the gene V protein-mediated control of gene II expression in bacteriophage M13 is an evolutionary relic of the ancestral filamentous-phage genome and thus dispensable for proper filamentous-phage replication.

Base Sequence↗
PubMed1997

In situ aggregational state of M13 bacteriophage major coat protein in sodium cholate and lipid bilayers.

The in situ aggregational behavior of the bacteriophage M13 major coat protein was determined for the protein isolated in sodium cholate and reconstituted into DOPC lipid bilayers. For this purpose, the cysteine mutants A49C and T36C of the major coat protein were labeled with either a maleimido spin-label or a fluorescence label (IAEDANS). The steric restrictions sensed by the spin-label were used to evaluate the local protein conformation and the extent of protein-protein interactions at the position of the labeled residue. In addition, fluorescent labels covalently attached to the protein were used to determine the polarity of the local environment. The labeled coat protein mutants were examined under different conditions of protein association (amphiphile environment, ionic strength, temperature, and pH). The aggregational state of the major coat protein solubilized from the phage particle in sodium cholate was not dependent on the ionic strength, but was strongly dependent on cholate concentration and pH during sample preparation. At pH 7.0 and high sodium cholate concentration, the protein was in a dimeric form. The unusually strong association properties of the protein dimer in sodium cholate at pH 7.0 were attributed to the inability of sodium cholate to disrupt the strong hydrophobic forces between neighboring protein subunits in the phage particle. Such a "structural protein dimer" was, however, completely and irreversibly disrupted at pH 10.0. Qualitatively the same aggregational tendency was found upon changing the pH for the coat protein reconstituted in DOPC lipid bilayers. This reveals that the dimer disruption process is primarily a protein property, because there are no titratable groups on DOPC in the experimental pH range. The results are interpreted in terms of a model relating the protein aggregational state in the assembled phage to the protein aggregational behavior in sodium cholate and lipid bilayers.

Bacteriophage M13↗
PubMed1988

Deuterium nuclear magnetic resonance investigation of bacteriophage M13 coat protein in dimyristoylphosphatidylcholine liposomes using palmitic acid as a probe.

The effect of incorporation of various amounts of M13 bacteriophage coat protein on the bilayer order and acyl chain motion in dimyristoylphosphatidylcholine (DMPC) liposomes has been investigated using deuterium NMR of specifically deuterated palmitic acid as a bilayer probe, phosphorus NMR and additional spin-label electron spin resonance (ESR). The secondary structure of the M13 coat protein in these bilayers was determined from circular dichroism spectra. Phosphorus NMR spectra of the mixed liposomes are characteristic for DMPC organized in bilayers, also after incorporation of various levels of M13 protein. Circular dichroism spectra of the coat protein indicate that the protein conformation is predominantly a beta-structure (more than 75%). Various incorporation levels of M13 coat protein do not affect the order of the deuterium-labelled positions along the acyl chain at the carbon-2, 9 and 16 positions. In contrast, the spin-spin relaxation times decrease at higher protein levels, especially at the carbon-16 position. The spin-label ESR spectra of the same system using 14-doxylstearic acid as a label show a second, motionally restricted component, that is not observed by deuterium NMR. The NMR and ESR results are consistent with a model in which the fatty acid molecules are in a fast two-site exchange (at a rate of approx. 10(7) Hz) between the sites in the bulk of the lipid bilayer and the motionally restricted sites on the coat protein.

Capsid↗
PubMed1982

Bilayer acyl chain dynamics and lipid-protein interaction: the effect of the M13 bacteriophage coat protein on the decay of the fluorescence anisotropy of parinaric acid.

Nanosecond fluorescence polarization anisotropy decay is used to determine the effect of the bacteriophage M13 coat protein on lipid bilayer acyl chain dynamics and order. The fluorescent acyl chain analogues cis- and trans-parinaric acid were used to determine the rate and extent of the angular motion of acyl chains in liquid crystalline (39 degrees C) dimyristoylphosphatidylcholine bilayers free of coat protein or containing the coat protein at a protein:lipid ratio of 1:30. Subnanosecond time resolution was obtained by using synchrotron radiation as the excitation source for single photon counting detection. Previous measurements of Förster energy transfer from coat protein tryptophan to cis- or trans-parinaric acid have shown that these probes are randomly distributed in the bilayer with respect to the protein. The anisotropy decay observed for pure bilayers has the form of a rapid drop, followed by a nonzero constant region extending from roughly 3 ns to at least 12 ns. The magnitude of the anisotropy in the plateau region is simply related to the acyl chain order parameter. The effect of the M13 coat protein is to increase the acyl chain order parameter significantly while having only a small effect on the rate of angular relaxation. This behavior is rationalized in terms of a simple microscopic model. The order parameters for pure lipid and coat protein containing bilayers are compared to 2H-NMR values.

Coliphages↗
PubMed1988

[Subcloning of DNA fragments of the simian adenovirus SA7 oncogene in bacteriophage M13].

The XmaI/PstI and XmaI DNA fragments of adenovirus SA7 oncogene and the adjacent region (16.7% of the physical map of SA7 left end DNA) were recloned in M13 bacteriophages mp8 and mp9 in order to obtain the singlestranded fragments EIa and EIb from the DNA region of monkey adenovirus SA7 located on the recombinant plasmid pASP carrying the DNA APstI fragment including the adenovirus SA7 oncogene.

Adenoviridae↗
PubMed1989

Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers.

Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.

Bacteriophages↗
PubMed1994

Structure of a foreign peptide displayed on the surface of bacteriophage M13.

The use of filamentous bacteriophage M13 as a vehicle for display of foreign peptides and proteins provides a means for the construction of therapeutic, diagnostic and technological tools of broad utility. The usefulness of this technology is dependent on the ability of an inserted peptide to act as a ligand when fused to a structural protein. This, in turn, depends on the configuration in which the fused peptide is presented on the surface of the phage. X-ray diffraction from oriented fibers of three M13 strains with different sequences inserted near the amino terminus of the major coat protein (gp8) has been used to demonstrate that the inserts do not affect the helical symmetry of the phage particles. The structure of one insertion mutant (M13BOM2) was analyzed in detail. This strain contains the pentapeptide GQASG inserted between amino acids 4 and 5 of the major coat protein. Analysis of fiber diffraction from this strain was used to obtain its structure to 7 A resolution. Examination of the resulting electron density map indicated that the insert is presented in an extended conformation in a shallow groove between two alpha-helices on the surface of the virion. This arrangement is reminiscent of the presentation of peptides by major histocompatibility antigens. The extended conformation of the peptide provides substantial surface exposure and puts it in a favorable position to act as a ligand in a biochemical process. This form of presentation may contribute to the high immunogenicity observed for peptides inserted into the gene 8 product of M13. The length of the groove appears to correspond to the upper length limit observed when foreign peptides are fused to all copies of gp8.

Amino Acid Sequence↗
PubMed2006

Inhibition of bacteriophage m13 replication with esterified milk proteins.

Esterified milk proteins [methylated (Met) or ethylated (Et) alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and beta-casein (BCN)], unmodified native milk proteins, and native basic proteins (calf thymus histone and hen egg white lysozyme) were tested for their antiviral activity against the bacteriophage M13 and for their influence on its replication (except BCN). All esterified milk proteins showed an antiviral activity against the bacteriophage M13, proportional to the extent of esterification and, hence, to the increased basicity of the modified proteins. Antiviral activity of 100% Met-BLG disappeared after its pepsinolysis but not after its trypsinolysis. The antiviral activity of Met-BLG was much higher than that of native basic proteins (histone and lysozyme). One hundred percent Met-BLG and 73% Et-BLG inhibited the replication of bacteriophage M13 completely, whereas 60% Met-ALA inhibited phage replication partially. Calf thymus histone inhibited the replication of bacteriophage M13 at a lower extent (20%) than Met- and Et-BLG (100% inhibition). Protein concentration, pH, and concentration of the Escherichia coli culture in the preincubation medium of the virus were other factors influencing antiviral activity. Interactions of esterified proteins with the phage DNA (phenol extracted) followed the same pattern as observed during studies of the inhibition of the phage replication: Met-BLG > Et-BLG > or = Met-ALA.

Animals↗
PubMed1993

M13 bacteriophage displaying disulfide-constrained microproteins.

A display-phage library (TN2), displaying an 18-residue peptide fused to coat protein III, represents a collection of up to 8.55 x 10(6) peptides encoded by only 1.68 x 10(7) DNA sequences. Each displayed peptide has two fixed cysteine residues (allowing disulfide formation) and six variegated residues, four between the cysteines and one either side of the cysteines. Screening this library against streptavidin (Sv) and the anti-beta-endorphin monoclonal antibody, 3-E7, yielded phage displaying disulfide-constrained microproteins with sequences similar to those published for the linear-peptide display phage. Analysis of selected clones indicated that a disulfide bond is required for high-affinity binding to each of the target proteins. The microproteins selected for binding to Sv and 3-E7 show more stringent sequence specificity than do linear peptides selected for binding to the same targets.

Amino Acid Sequence↗
PubMed1998

Display of functional beta-lactamase inhibitory protein on the surface of M13 bacteriophage.

The display of proteins on the surface of filamentous phage has been shown to be a powerful method to select variants of a protein with altered binding properties from large combinatorial libraries of mutants. The beta-lactamase inhibitory protein (BLIP) is a 165-amino-acid protein that binds and inhibits TEM-1 beta-lactamase-catalyzed hydrolysis of the penicillin and cephalosporin antibiotics. Here we describe the construction of a new phagemid vector and the use of this vector to display BLIP on the surface of filamentous phage. It is shown that BLIP-displaying phage bind to immobilized beta-lactamase and that the binding can be competed off by the addition of soluble beta-lactamase. In addition, a two-step phage enzyme-linked immunosorbent assay procedure was used to demonstrate that the BLIP-displaying phage bind beta-lactamase with a 50% inhibitory concentration of 1 nM, which compares favorably with a previously published Ki of 0.6 nM. A system has therefore been established for protein engineering of BLIP to expand its range of binding to other beta-lactamases and penicillin-binding proteins.

Amino Acid Sequence↗