PubMed · 8959715
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D Dottavio. 1996. Epitope mapping using phage-displayed peptide libraries.. https://doi.org/10.1385/0-89603-375-9%3A181
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A synthetic gene for hirudin was ligated into phagemid pCANTAB5E. This construct allows production of either soluble hirudin or phage having hirudin displayed on the surface. Similarly, hirudin variants with extensions either at their N- or C-terminus were generated. The genes were expressed in their soluble form in a non-suppressor strain of E. coli. Periplasmatic fractions were evaluated in standard thrombin inhibition assays. Extending hirudin by a single Gln residue at the N-terminus reduces the activity by two orders of magnitude. This suggests that either the terminal amine group makes an important interaction or that steric constraints do not allow additional amino acids here. Only C-terminal extensions maintain most of the thrombin inhibitor activity of r-hirudin. The r-hirudin gene was also expressed on the tips of filamentous phage as a fusion protein with protein III (pIII). The hirudin-pIII fusion protein was detected with anti-hirudin antibody and with anti-E-tag antibody by Western blot analysis. Recombinant phages were shown to bind to immobilized thrombin in a dose-dependent manner. Upon addition of soluble thrombin, recombinant hirudin phages could be eluted specifically. Finally, purified phages carrying displayed r-hirudin were shown to inhibit thrombin in a standard amidolytic assay for thrombin inhibitor activity. These results demonstrate that hirudin can be C-terminally extended without diminishing the antithrombic activity. Beyond that, active hirudin can be displayed on the surface of M13 phage. As a conclusion, applied molecular evolution, i.e. the selection of hirudin-based thrombin inhibitor variants with tailored properties from (partially) randomized peptide pools should now be possible.
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.
The gene 59 protein (gp59) of bacteriophage T4 is an important accessory protein of the phage-encoded replicative DNA helicase, gp41. The properties of this 26 kDa protein include selective binding to ssDNA, and specific interactions with both gp41 and gp32, the T4-encoded ssDNA- binding protein. gp59 stimulates many of the DNA-dependent activities of the gp41 enzyme by promoting its assembly onto gp32-ssDNA complexes. Direct interactions between gp59 and gp32-ssDNA complexes are essential for helicase assembly, and gp59-gp32 protein-protein interactions have been shown to play a central role. Presumably, the ssDNA-binding activity of gp59 is also important for helicase assembly; however, to date this activity has been poorly characterized. In this study, we present the first detailed biochemical investigation of the interactions of gp59 with single-stranded polynucleotides. Using etheno-DNA fluorescence enhancement and quantitative ssDNA-cellulose methods, we demonstrate the following: (1) gp59 binds to single-stranded polynucleotides with a binding site size of nine to ten nucleotide residues per monomer; (2) gp59 exhibits relative affinities towards four different ssDNA lattices used in this study according to the heirarchy: ssDNA (random sequence) > epsilonDNA (random sequence) > poly(dA) > poly(depsilonA); (3) gp59 exhibits two or more different polynucleotide binding modes distinguished by their cooperativities of binding, and modulated by salt and/or lattice effects; (4) gp59-ssDNA binding is characterized by a large salt effect on the association constant, consistent with multiple ionic contacts between protein and ssDNA phosphate residues and with the displacement of anions from the protein. The implications of our findings for the mechanism of action of gp59 in helicase-ssDNA assembly are discussed.