Phage display

The phage display (English phage display ) is a biotechnological method when of large recombinant libraries peptides, protein parts (eg antibody fragments) or complete proteins are functionally displayed on the surface of bacteriophages to then suitable binding partner for a particular ligand to isolate and identify. The phage display is for the elucidation of protein- protein interactions for the development of new biological drugs and to search for specific antibodies for therapeutic, diagnostic or experimental applications of great importance.

The method was introduced as the first form of a molecular display technology by George P. Smith 1985.

Principle

The principle of molecular displays based on the common occurrence of a protein and its encoding DNA in a particle (in this case there are bacteriophage ), which on the basis of binding to an already present protein (or other molecule ) an interaction partner consisting of a mixture of transgenic bacteriophage can be isolated, the DNA is then present as well. The recombinant surface protein corresponding DNA sequence is then extracted, copied by PCR and sequenced by DNA sequencing. Via the genetic code, then the amino acid sequence of a binding protein is known.

The phage display respectively a plurality of any DNA sequences in the DNA sequence of an envelope protein in the genome of the bacteriophage or phagemid are generally parallel ligated so that the information encoded in the sequence of the proteins or peptides of the N-terminus as a fusion protein on the surface of bacteriophage are presented. The presentation of the protein on the surface allows for selection of phage by affinity for a particular molecule. Thereby can be produced and isolated which are capable of binding, due to their fusion protein to a molecule for which an interaction partner is being sought for any mixture of DNA sequences, recombinant bacteriophage.

Phage display can with filamentous phage (e.g., f1 phage, M13 phage or fd phage ) with T4 and T6 phage ( Myoviridae ), with λ phage ( Siphoviridae ) or T7 phages ( Podoviridae ) are performed. Accordingly, the protein can be at most filamentous phage g3p ( pIII synonymous, rarely also pVI, pVII, pVIII or pIX is used) as a fusion partner uses on the virus surface, in phage T4 Soc and Hoc are mostly used as a fusion protein.

The assembly of the bacteriophage is carried out in bacteria. Viral membrane proteins must first be stored for assembly of a filamentous phage in the bacterial cell membrane in order to aggregate there with the capsid. In bacteria, there are three systems of this secretion of membrane proteins, the Sec - system, the SRP - system and the TAT system. The use of lytic phages, such as T4, T6, T7, or λ - phage, however, does not require incorporation of viral proteins in the cell membrane.

The Sec - and SRP system unfold a protein translocation across the cell membrane. The TAT system is however easier to saturate, but it can inject folded proteins of up to 180 kilodaltons through the membrane. In addition, the TAT system, the protein folding take place already in the reducing environment of the cytosol, which may be necessary for cytosolic proteins for proper folding, because outside of the cell membrane ( the periplasm ) can form undesired disulfide bonds that may prevent proper folding. In the lytic phage there is no problem with disulfide -induced protein misfolding as they are assembled in the cytosol, where disulfide bonds can not occur due to the reducing environment.

Phage display of antibody libraries

First antibody-producing B cells ( plasma cells) are isolated from the blood, bone marrow or lymph nodes of a dispenser. From the mRNA is recovered and transcribed into cDNA. Using the polymerase chain reaction ( PCR), the genes of the light (VL) and heavy chain (VH ) of the antibody can be amplified from the cDNA. Each of genes ( VH and VL ) is connected to the truncated gene of the coat protein pIII (minor coat protein ) of the M13 phage was ligated into a specific phagemid vector and Escherichia coli transformed therewith. Thereby expressing E. coli bacteria pIII fusion proteins of scFv fragments or Fab antibody fragments. The fusion proteins are indicated by a signal peptide ( pelB or ompA arise in ) transported into the periplasm, where they fold into a functional scFv or Fab fragment bandaged by disulfide bridge. The Fv or Fab shares initially remain anchored over the pIII fragment into the inner E. coli membrane and bind upon completion of the assembly of the phage to the capsid.

On the coat protein pIII, is normally responsible for the infection of the bacteria, after co-infection with M13 helper phage ( for the expression of the non-modified pIII and the other phage proteins ) incorporated the functional antibody fragment, the maturation of newly formed phages in the outer shell. Simultaneously, the phagemid with the corresponding genetic information for the corresponding antibody fragment is incorporated into the interior of the newly formed phage. Each of these recombinant phages has theoretically a different antibody fragment on its surface and at the same time the corresponding genes ( VH and VL ) in its interior, similar to the billions of B cells in the (human) body.

In a so-called biopanning, the " binding " can be fished out of the phages exposed on the surface of antibody fragments by interaction with fixed ligands ( antigens ) from the billions of background the irrelevant phage ( gene library ).

From the thus isolated " monoclonal " antibody phage, the associated antibody genes can be easily isolated and sequenced. Also, can thus be produced as soluble proteins for specific applications in mass culture in E. coli or other cell systems, the selected antibody fragments.

Phage display peptide libraries

In the same manner as with antibodies, it is possible to select the peptide sequences ( 9-12 amino acid residues ) on the basis of their affinity for specific target molecules.

This technique is useful to select small molecules that are easier than larger molecules such as drugs which may later be used, for example, antibodies or other proteins.

Another application of peptide libraries is also the determination of epitope for monoclonal antibodies.

Phage Display of cDNA libraries

Hitherto little used is also the preparation of cDNA expression libraries in phage. Since frequently occurring stop codons in the sequences of many libraries, it is not possible to combine the sequences of the library prior to the pIII protein of filamentous phage.

Therefore be used as an alternative lytic phage or the body designated as Jun / Fos- system non-covalent interaction of the storage protein from the inserted DNA sequence and pIII of filamentous phage. In the Jun / Fos- jun system initially linked to the pIII protein. On the same phagemid Fos is placed before the sequences to be expressed. Since both proteins are present on a phagemid, both proteins are expressed at the same time, Jun and Fos dimerize through their leucine zipper and coupled in this way, the pIII protein of the expressed cDNA sequence.

Applications and perspectives

The phage display is a technique, which is based on protein-protein interactions, and therefore is suitable as a method for detection of interactions between proteins. Display techniques have allowed for the first time the production and characterization of many new human antibodies. Other proteins, for example in the form of cDNA libraries can be selected in phage display. The affinity of the selected antibody can be increased by mutagenesis. In recent years there have been many improvements in the techniques, but phage display is far from routine. Several companies now offer to produce very large, partly semi-synthetic phage display libraries antibodies against virtually any antigen. Recombinant antibodies account for about 30% of all currently in clinical testing biopharmaceuticals; This shows the potential for growth of biotechnology is in the production of these products.