System comprising bacteriophages and particles that contain active substances

Abstract

The present invention concerns a system, comprising bacteriophages and particles comprising active agents, in which a first additional peptide is fused to proteins of the bacteriophage, the first additional peptide adheres to the surface of the particle and furthermore a second additional peptide is fused to proteins of the bacteriophage. The second additional peptide can adhere on substrate surfaces. The present invention furthermore concerns the use of the system for delayed release of active agents and also a method for production of the system. The present invention furthermore concerns a method for the selection of phage species from a combinatorial phage population.

1 . Method for the selection of phage species from a combinatorial phage population, comprising the following steps:
 a. exposing an A Phage display library to a substrate in a buffered aqueous environment,   b. washing the substrate,   c. detaching the binding phages using an aqueous elution buffer,
 wherein the washing of the substrate takes place with energy input by means of ultrasonics. 
   
     
     
         2 . Method for the production of a system comprising a bacteriophage and a particle comprising an active agent, wherein a first additional peptide is fused to a protein of the bacteriophage, the first additional peptide adheres to a surface of the particle and furthermore a second additional peptide is fused to a protein of the bacteriophage, said method comprising providing a phage clone of type M13, preselected by panning, with a first additional peptide already fused to gpIII, which imparts binding properties to an active agent particle, and fusing a second additional peptide to the gpVIII protein of the same phage clone, with it being possible for the peptides fused to gpIII and gpVIII to be the same or different. 
     
     
         3 . Method according to  claim 2 , comprising the following steps:
 a. introducing restriction cleavage sites into the gpVIII procoat sequence, by carrying out localized mutagenesis on the gpVIII procoat gene, which is located on a plasmid between two suitable restriction cleavage sites,   b. transferring the mutations from step a) by recloning from the plasmid into the replicative form (RF) of the M13 genome,   c. cleaving the replicative form of the phage DNA with those restriction enzymes whose cleavage sites were introduced by the mutations from step 1), and then serves to assimilate suitable complementary oligonucleotides which at the ends show the corresponding overhang for ligation into the open restriction sites and which code for a second additional peptide sequence.   
     
     
         4 . Method according to  claim 3 , wherein the restriction cleavage sites are an NcoI cleavage site in the signal sequence of gpVIII and an PstI cleavage site in the mature region. 
     
     
         5 . Method for the production of a system comprising a bacteriophage and a particle comprising an active agent, wherein a first additional peptide is fused to a protein of the bacteriophage, the first additional peptide adheres to a surface of the particle and furthermore a second additional peptide is fused to a protein of the bacteriophage, said method comprising providing a phage clone of type M13, preselected by panning, with a first additional peptide already fused to gpIII, and fusing a second additional peptide which imparts binding properties to an active agent particle to the gpVIII protein of the same phage clone, with it being possible for the peptides fused to gpIII and gpVIII to be the same or different. 
     
     
         6 . Method of conferring adherence, comprising conferring adherence with a peptide of the sequence ISSKPTSQLTT-spacer-PSTTRLR (SEQ ID NOS: 10-18), where the spacer comprises ≧0 to ≦10 repeating units of the amino acids glycine and/or alanine.