Nucleotide sequences in bacteriophage f1 DNA: nucleotide sequence of genes V, VII, and VIII.

The sequence of nucleotides comprising genes V, VII, and VIII of bacteriophage f1 was determined. The sequence was found to differ from that of the corresponding region of the related fd genome by eight base substitutions in gene V and one in gene VIII. The structure of gene VII was completely conserved between these two viruses and was identical to that of bacteriophage M13. Both transitions and transversions were found in cases where bases were substituted, but all substitutions were in the third codon position and had no effect on the structure of the corresponding protein product. The gene V protein product could thus be deduced to be identical to that of the corresponding proteins from bacteriophages fd and M13. A potential EcoRII cleavage site was formed by nucleotides 172 to 176 of gene V. Replicative form DNA form DNA from bacteriophage f1 is normally resistant to this enzyme, and evidence is presented to suggest that the sequence was modified through methylation of cytosine 173. The probable locations of other modified nucleotides in the sequence are discussed.     

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.     

New vectors for peptide display on the surface of filamentous bacteriophage

We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion. These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits.     

Variable electrostatic interaction between DNA and coat protein in filamentous bacteriophage assembly

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the DNA of the class I filamentous bacteriophage fd, which infects Escherichia coli. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter, generating plasmid pKf8P. Bacteriophage fd gene VIII was similarly cloned into the plasmid pEMBL9+, enabling it to be subjected to site-directed mutagenesis. By this means the positively charged lysine residue at position 48, one of four positively charged residues near the C terminus of the protein, was turned into a negatively charged glutamic acid residue. The mutated fd gene VIII was cloned back from the pEMBL plasmid into the expression plasmid pKK223-3, creating plasmid pKE48. In the presence of the inducer isopropyl-beta-D-thiogalactoside, the wild-type and mutated coat protein genes were strongly expressed in E. coli TG1 cells transformed with plasmids pKf8P and pKE48, respectively, and the product procoat proteins underwent processing and insertion into the E. coli cell inner membrane. A net positive charge of only 2 on the side-chains in the C-terminal region is evidently sufficient for this initial stage of the virus assembly process. However, the mutated coat protein could not encapsidate the DNA of bacteriophage R252, an fd bacteriophage carrying an amber mutation in its own gene VIII, when tested on non-suppressor strains of E. coli. On the other hand, elongated hybrid bacteriophage particles could be generated whose capsids contained mixtures of wild-type (K48) and mutant (E48) subunits. This suggests that the defect in assembly may occur at the initiation rather than the elongation step(s) in virus assembly. Other mutations of lysine-48 that removed or reversed the positive charge at this position in the C-terminal region of the coat protein were also found to lead to the production of commensurately longer bacteriophage particles. Taken together, these results indicate direct electrostatic interaction between the DNA and the coat protein in the capsid and support a model of non-specific binding between DNA and coat protein subunits with a stoicheiometry that can be varied during assembly.     

Peptides derived from a secretory yeast library restore factor VIII activity in the presence of an inhibitory antibody

The development of autoantibodies against factor VIII represents one of the major complications in the treatment of hemophilia A patients. We have employed a novel library system to obtain peptides that specifically neutralize the interaction between factor VIII and these inhibitors. The random peptides are presented as carboxy-terminal extensions of the eukaryotic initiation factor 5a, an intracellular protein with a molecular mass of 18 kDa. These random peptides formed an unique binding site, as demonstrated by molecular simulations using the computer programs InsightII and GROMACS. The library was screened to identify peptides binding to the murine monoclonal anti-factor VIII antibody ESH8 and to inhibitors derived from patients with factor VIII antibodies. Ten peptides binding to ESH8 were identified. Their specificity was confirmed by displacement assays. Two peptides with the sequences STKTLGRPLHGPAGPVEGGALAGVAEDADLVTAVSGR and YHCKREDLTDRDATCALRQPPQAVRGLGPRVTAVSGR showed the ability to restore the factor VIII activity from 33% up to approximately 90% in functional tests performed in vitro. Three candidates for binding to factor VIII antibodies derived from four different patient's sera were achieved. Three fusion proteins with the peptide sequences PQLGSRRSTTPSLTFQNASWFPAGGPCARSNRG, SGSRQVCKLARSLQPF and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP bound to inhibitors derived from different patients. Furthermore, two of the obtained fusion proteins with the peptide sequences RHWTALGPAPTHTCADLNYPLLS and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP did also bind to the monoclonal antibody ESH8. This study demonstrates the potential of this system to identify peptides that inhibit the activity of potent inhibitory antibodies and also shows potential as a method for screening of bioactive peptides.     

Effects of bacteriophage f1 gene III protein on the host cell membrane

Summary Plasmids which encode bacteriophage f1 coat protein genes VIII and III are responsible for a number of unusual properties suggesting that they have a drastic effect on the bacterial outer membrane. Analysis of several such recombinant plasmids and selection of mutant plasmids unable to cause this effect established that the properties were caused by gene III protein or its amino-terminal fragment.     

Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens

We describe here two systems for encoding foreign amino acid sequences in the exposed N-terminal segment of the major coat protein of bacteriophage fd. Small peptides can be encoded directly; larger peptides are encoded in hybrid bacteriophage particles, in which the capsid is formed from a mixture of wild-type and modified coat proteins. In both cases, the peptides are present in multiple copies per phage particle. Peptides that represent the circumsporozoite protein, the major surface antigen of the sporozoites of the malaria parasite, Plasmodium falciparum, were inserted in this way and found to be highly immunogenic. These systems should prove to be valuable in displaying specific or random peptides as antigens, and could lead to cheap and effective vaccines. They will also allow rapid screening of peptides as potential agents of other biological effects, with important applications in biomolecular design.     

A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda

Bacteriophage lambda (λ) permits the display of many foreign peptides and proteins on the gpD major coat protein. However, some recombinant derivatives of gpD are incompatible with the assembly of stable phage particles. This presents a limitation to current λ display systems. Here we describe a novel, plasmid-based expression system in which gpD deficient λ lysogens can be co-complemented with both wild-type and recombinant forms of gpD. This dual expression system permits the generation of mosaic phage particles that contain otherwise recalcitrant recombinant gpD fusion proteins. Overall, this improved gpD display system is expected to permit the expression of a wide variety of peptides and proteins on the surface of bacteriophage λ and to facilitate the use of modified λ phage vectors in mammalian gene transfer applications.     

Topography of the human factor VIII-von Willebrand factor complex

Factor VIII circulates in noncovalent complex with von Willebrand factor (vWf). The topography of this complex was evaluated by fluorescence energy transfer using factor VIII subunits modified with N-(1-pyrenyl)maleimide (NPM; fluorescence donor) and vWf-derived fragments modified with 7-diethylamino-3-[4'-maleimidylphenyl]-4-methyl coumarin (CPM; fluorescence acceptor). Results from a previous study indicated an interfactor VIII subunit distance of 20 A separating Cys528 and Cys1858 in the factor VIII heavy and light chains, respectively (Fay, P.J., and Smudzin, T. M. (1989) J. Biol. Chem. 264, 14005-14010). Fluorophore modification of the vWf SPIII homodimer (residues 1-1365) indicated multiple attachment sites at Cys126/135/1360 as determined from sequence analysis of fluorescent tryptic peptides derived from the modified protein. Based upon donor quenching data, an interfluorophore distance of approximately 28 A was calculated separating NPM-factor VIII light chain or factor VIII reconstituted from NPM-light chain plus unmodified heavy chain, from CPM-SPIII. A similar value (29 A) was obtained for NPM-light chain paired with CPM-SPIII-T4 (vWf residues 1-272), suggesting that donor quenching resulted primarily from modified residue(s) Cys126/135 in the acceptor. No energy transfer was observed for the NPM-heavy chain/CPM-SPIII pairing. However, when NPM-heavy chain was reassociated with unmodified light chain prior to reaction with CPM-SPIII or CPM-SPIII-T4, energy transfer was observed with calculated interfluorophore distances of approximately 31 and 34 A, respectively. Levels of acceptor resulting in maximal donor quenching suggested an equimolar stoichiometry of factor VIII (light chain)/vWf fragment in the reconstituted complexes. These results indicate a close spatial arrangement among the A3 domain of factor VIII light chain, the A2 domain of factor VIII heavy chain, and the NH2 terminus region of vWf in the factor VIII-vWf complex.     

Intersubunit fluorescence energy transfer in human factor VIII

Human factor VIII circulates as a series of active heterodimers composed of a light chain (83 kDa) linked by divalent metal ion(s) to a variable sized heavy chain (93-210 kDa). Purified factor VIII subunits were modified with sulfhydryl-specific fluorophores. Probe selection was based upon the limited number of free cysteine residues in each subunit. Levels of probe incorporation suggested the presence of a single reactive cysteine residue per subunit. Amino-terminal sequence analysis of fluorescent tryptic peptides derived from the modified subunits indicated fluorophore attachment sites at Cys528 of the heavy chain (A2 domain) and Cys1858 of the light chain (A3 domain). Subunit reassociation was measured by fluorescence energy transfer using light chain modified with N-[1-pyrenyl] maleimide (fluorescence donor) and heavy chain modified with 7-diethylamino-3-[4'-maleimidophenyl]-4-methylcoumarin (fluorescence acceptor). Donor fluorescence quenching paralleled the formation of factor VIII clotting activity, and both effects were saturable with respect to added heavy chain. Based upon the degree of donor quenching, a distance of 20 A was calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A3 domain of light chain in the factor VIII heterodimer.     

A set of expression plasmids for the synthesis of fused and unfused polypeptides in Escherichia coli

A set of plasmid expression vectors for cloning of DNA fragments containing open reading frames has been obtained. The plasmids carry the strong leftward promoter of bacteriophage lambda and the translation initiation signals from either the gene ner of bacteriophage Mu or the gene 4 of bacteriophage phi 29. The vectors could overexpress the cloned sequences as fusion peptides at the N terminus with the N-terminal segment of the phi 29 protein p4 or at the C terminus with the Escherichia coli beta-galactosidase from its 8th residue, or both. Alternatively, the cloned sequences could be directed to overproduce proteins in an unfused form. DNA fragments of the hemagglutinin gene from human influenza A virus, have been cloned in one of the plasmid vectors and some potential antigenic determinants have been characterized using monoclonal antibodies.     

The DNA-binding protein of Pf1 filamentous bacteriophage: amino-acid sequence and structure of the gene

The amino-acid sequence of the single-stranded DNA-binding protein of bacteriophage Pf1 and the nucleotide sequence of the corresponding gene have been determined. The protein has 144 amino acids and a molecular weight of 15 400; the gene consists of 435 nucleotides. The amino-acid sequence was determined by Edman degradation, carboxypeptidase A, B, and P digestion of intact protein and of peptides derived by chymotrypsin, Staphylococcus aureus V8 protease, and trypsin digestion. The nucleotide sequence was determined by the dideoxy method after random cloning of fragments of Pf1 DNA into M13. No sequence homology could be established between the amino-acid sequence of the DNA-binding protein of Pseudomonas aeruginosa-specific bacteriophage Pf1 and bacteriophage fd of Escherichia coli.     

Structure-Activity Relationships of Vasopressin Analogues on Release of Factor Viii in Dogs

Abstract

In order to study structure-activity relationships as to Factor VIII release conscious dogs were injected with analogues of vasopressin. The peptides used were chemically modified either in the hexapeptide ring structure of the vasopressin molecule or in the C-terminal tripeptide or in both. The results showed that an intact C-terminal appears to be of importance for retaining Factor VIII releasing activity of the analogues, whereas at least some modifications of the ring structure are tolerated without loss of activity. Decreased activity was also observed when the disulphide bridge was substituted with a monocarba bond.     

A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery

There is considerable interest in the use of bacteriophage vectors for mammalian cell gene transfer applications, due to their stability, excellent safety profile and inexpensive mass production. However, to date, phage vectors have been plagued by mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian cells and the need to refine each step of this process in order to arrive at an optimal, phage-based gene transfer system. Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacteriophage lambda. Using this novel method, multiple peptides were displayed simultaneously from both the phage head and tail. Surface head display of an ubiquitinylation motif greatly increased the efficiency of phage-mediated gene transfer in a murine macrophage cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding motif. Overall, this work provides a novel system that can be used to rationally improve bacteriophage gene transfer vectors and shows it may be possible to enhance the efficiency of phage-mediated gene transfer by targeting and optimizing multiple steps within the viral infection pathway.     

Peptide affinity chromatography of human clotting factor VIII: Screening of the vWF-binding domain

The region of von Willebrand factor, which is involved in the complex formation with factor VIII, was used to generate a panel of octapeptides. A peptide ladder was generated from the von Willebrand factor region aa40 to aa100 and was synthesized on cellulose membranes by spot technology. Four peptides with affinity for factor VIII were identified by incubation with plasma derived factor VIII and recombinant factor VIII. The peptides denoted as 010 (LCPPGMVRHE), 011 (RCPCFHQGK), 014 (CFHQGKEYA) and 015 (RDRKWNCTDHVC) were further characterized by real-time interaction analysis and small scale affinity chromatography. Biotinylated peptides were used for blotting assays. These experiments showed that the peptides are directed against the light chain of FVIII. We consider these peptides as valuable tools for in situ labeling and also as ligands suitable for affinity chromatography.