Phage display

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The phage display ( english phage display ) is a biotechnological method of large, in the recombinant libraries peptides (z. B., protein portions antibody fragments ) or complete proteins functionally on the surface of bacteriophage are presented, specific in order to then suitable binding partner for an Isolate and identify ligands . The phage display is of great importance for the elucidation of protein-protein interactions , for the development of new biological drugs and for the search for specific antibodies for therapeutic, diagnostic or experimental applications.

The method was introduced by George P. Smith in 1985 as the first form of molecular display technique . He received the Nobel Prize for Chemistry in 2018 together with Greg Winter , who further developed the technology so that parts of human antibodies were also presented in the phage display.

principle

General scheme of in vitro biopanning as a central element of the phage display

The principle of the molecular display is based on the common occurrence of a protein and its coding DNA in a particle (in this case bacteriophages ), whereby an interaction partner from a mixture of transgenic bacteriophages based on the binding to an already existing protein (or other molecule) can be isolated whose DNA is then also available. The DNA sequence corresponding to the recombinant surface protein is then extracted, amplified by PCR and sequenced by DNA sequencing . The amino acid sequence of the binding protein is then also known via the genetic code .

In the case of phage display, several arbitrary DNA sequences are usually ligated in parallel to the DNA sequence of an envelope protein in the genome of the bacteriophage or in a phagemid , so that the proteins or peptides encoded in this sequence are N-terminally as a fusion protein on the surface of the bacteriophage to get presented. The presentation of the proteins on the surface allows a selection of the phages to the affinity to a specific molecule. As a result, recombinant bacteriophages can be generated and isolated from any mixtures of DNA sequences which, due to their fusion protein, can bind to a molecule for which an interaction partner is being sought.

The phage display can be carried out with filamentous phages (e.g. f1 phages , M13 phages or fd phages ), with T4 and T6 phages ( Myoviridae ), with λ phages ( Siphoviridae ) or with T7 phages ( Podoviridae ). Accordingly, with filamentous phages, the protein g3p (synonym pIII, pVI, pVII, pVIII or pIX is also used more rarely) as a fusion partner on the virus surface, with T4 phages , Soc or Hoc are usually used as the fusion protein.

The assembly of the bacteriophages takes place in bacteria . In order to assemble a filamentous phage, viral membrane proteins first have to be embedded in the bacterial cell membrane in order to be attached to the capsid there . In bacteria there are three systems for the 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 phages, on the other hand, does not require the storage of viral proteins in the cell membrane.

The Sec and SRP systems unfold a protein for translocation across the cell membrane. The TAT system, on the other hand, is easier to saturate , but it can move folded proteins of up to 180  kilodaltons across the membrane. In addition, in the TAT system, protein folding can already take place in the reducing environment of the cytosol , which can be necessary for correct folding of cytosolic proteins, because unwanted disulfide bridges can form outside the cell membrane (in the periplasm ) , which can prevent correct folding. With the lytic phages there is no problem with protein misfolding caused by disulfide bridges, since they are assembled in the cytosol, where no disulfide bridges can arise due to the reducing environment.

Phage display from antibody libraries

First, antibody-producing B cells (plasma cells) are isolated from the blood, bone marrow or lymph nodes of a donor. The mRNA is obtained from this and transcribed into cDNA . With the aid of the polymerase chain reaction (PCR), the genes of the light (V L ) and heavy chain (V H ) of the antibodies are multiplied from the cDNA . Each set of genes (V L and V H ) is ligated with the shortened gene of the coat protein pIII ( minor coat protein ) of the M13 phage in a special phagemid vector and Escherichia coli is transformed with it . As a result, the E. coli bacteria express pIII fusion proteins with scFv fragments or Fab antibody fragments . The fusion proteins are transported into the periplasm by a signal peptide (derived from pelB or ompA ) , where they fold into a functional scFv or Fab fragment linked by a disulfide bridge. The Fv and Fab parts initially remain anchored in the inner E. coli membrane via the pIII fragment and bind to the capsid when the assembly of the phage is complete.

After co-infection with an M13 helper phage (for the expression of the unmodified pIII and the other phage proteins), the functional antibody fragment is incorporated into the outer envelope of newly formed phages during the maturation process via the coat protein pIII, which is normally responsible for the infection of the bacteria. At the same time, the phagemid with the associated genetic information for the corresponding antibody fragment is incorporated into the interior of the newly formed phage. In theory, each of these recombinant phages has a different antibody fragment on its surface and at the same time the associated genes (V L and V H ) inside, comparable to the billions of B cells in the (human) body.

In a so-called biopanning , the “binding” phages can be fished out of the billions of times the background of the irrelevant phages ( gene library ) via the antibody fragments exposed on the surface through interaction with fixed ligands ( antigens ) .

The associated antibody genes can be easily isolated and sequenced from the “monoclonal” antibody phages isolated in this way. The selected antibody fragments can also be produced as soluble proteins for special applications in mass culture in E. coli or other cell systems.

Phage display from peptide libraries

In the same way as with antibodies, it is possible to select peptide sequences (9 to 12 amino acid residues ) based on their affinity for certain target molecules.

This technique is useful to select small molecules that later prove to be easier drugs than larger molecules such as B. use antibodies or other proteins.

Another possible application for peptide libraries is the determination of epitope sequences in monoclonal antibodies.

Phage display from cDNA libraries

The representation of cDNA expression libraries on phages has also not been used much so far . Since stop codons often occur in the sequences of many libraries , it is not possible to join the library sequences in front of the pIII protein of filamentous phages.

Therefore, as an alternative, lytic phages or the non-covalent assembly of the protein from the inserted DNA sequence and pIII from filamentous phages, referred to as the Jun / Fos system , are used. In the Jun / Fos system, Jun is first coupled to the pIII protein. On the same phagemid , Fos is placed in front of the sequences to be expressed . Since both proteins are contained on a phagemid, both proteins are expressed at the same time, Jun and Fos dimerize via their leucine zipper and in this way couple the pIII protein to the expressed cDNA sequence.

Applications and perspectives

Phage display is a technique that is based on protein-protein interactions and is therefore suitable as a method for detecting interactions between proteins. Display techniques made it possible for the first time to produce and characterize many new human antibodies. Other proteins, e.g. B. in the form of cDNA libraries, can be selected in the phage display. The affinity of the selected antibodies can be increased by mutagenesis. There have been many improvements in techniques in recent years, but phage display is far from being routine. Several companies are now offering, from very large, e.g. T. semisynthetic phage display libraries produce antibodies against virtually any antigen. Recombinant antibodies make up about 30% of all biopharmaceuticals currently in clinical testing ; this shows the potential for the growth of biotechnology in the production of these products.

Individual evidence

  1. ^ GP Smith: Filamentous Fusion Phage: Novel Expression Vectors That Display Cloned Antigens on the Virion Surface . In: Science . tape 228 , no. 4705 , June 14, 1985, pp. 1315-1317 , doi : 10.1126 / science.4001944 .
  2. ^ A b c W. Li, NB Caberoy: New perspective for phage display as an efficient and versatile technology of functional proteomics. In: Appl Microbiol Biotechnol. (2010), Volume 85 (4), pp. 909-19. PMID 19885657 ; PMC 2992952 (free full text).
  3. ↑ JW Kehoe, BK Kay: Filamentous phage display in the new millennium. In: Chem. Rev. (2005), Vol. 105 (11), pp. 4056-72. PMID 16277371 .
  4. ^ VB Rao, LW Black: Structure and assembly of bacteriophage T4 head. In: Virol J. (2010), Volume 7, p. 356. PMID 21129201 ; PMC 3012670 (free full text).
  5. ↑ N. Velappan, HE Fisher, E. Pesavento, L. Chasteen, S. D'Angelo, C. Kiss, M. Longmire, P. Pavlik, AR Bradbury: A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins. In: Nucleic Acids Res . (2010), Volume 38 (4), p. E22. PMID 19955231 ; PMC 2831335 (free full text).
  6. ↑ J. Speck, KM Arndt, KM Müller: Efficient phage display of intracellularly folded proteins mediated by the TAT pathway. In: Protein Eng Des Sel. (2011), Volume 24 (6), pp. 473-84. PMID 21289038 . PDF .
  7. ↑ J. Rakonjac, NJ Bennett, J. Spagnuolo, D. Gagic, M. Russel: Filamentous bacteriophage: biology, phage display and nanotechnology applications. In: Curr Issues Mol Biol. (2011), Volume 13 (2), pp. 51-76. PMID 21502666 . PDF .
  8. ↑ B. Braun, M. Paschke: Phage display on new ways . In: Biospectrum . 12, No. 4, 2006, pp. 381-383.
  9. ↑ Reto Crameri, Rolf Jaussi, Gunter Menz, Kurt Blaser: Display of Expression Products of cDNA libraries on phage Surfaces . In: European Journal of Biochemistry . tape 226 , no. 1 , November 1, 1994, pp. 53-58 , doi : 10.1111 / j.1432-1033.1994.00t53.x .
  10. ↑ Sachdev S Sidhu, Wayne J Fairbrother, Kurt Deshayes: Exploring Protein-Protein Interactions with Phage Display . In: ChemBioChem . tape 4 , no. 1 , January 3, 2003, p. 14-25 , doi : 10.1002 / cbic.200390008 .

literature

  • Thomas Schirrmann, Michael Hust, Stefan Dübel: The antibody factory: Antibodies for every protein . In: Biology in Our Time . tape 37 , no. 6 , December 1, 2007, p. 348–351 , doi : 10.1002 / biuz.200790096 .
  • Hennie R. Hoogenboom: Selecting and screening recombinant antibody libraries . In: Nature Biotechnology . tape 23 , no. 9 , September 7, 2005, pp. 1105-1116 , doi : 10.1038 / nbt1126 (review).
  • A. Schmiedl, S. Dübel: Recombinant Antibodies & Phage Display In: M. Wink. (Ed.): Molecular Biotechnology. Wiley-VCH. 2004.
  • Valery A Petrenko, Iryna B Sorokulova: Detection of biological threats. A challenge for directed molecular evolution . In: Journal of Microbiological Methods . tape 58 , no. 2 , August 2004, p. 147-168 , doi : 10.1016 / j.mimet.2004.04.004 .
  • Peter J. Hudson, Christelle Souriau: Engineered antibodies . In: Nature Medicine . tape 9 , no. 1 , January 1, 2003, p. 129-134 , doi : 10.1038 / nm0103-129 (review).
  • R. Konterman, S. Dübel: Antibody Engineering - Springer Lab Manual Springer, Heidelberg 2001.
  • F. Breitling, S. Dübel: Recombinant Antibodies. , Spektrum Akad., Heidelberg 1997.
  • P. Fischer: Expression of the human antibody repertoire with bacteriophages: Techniques, applications and perspectives. In: Biospectrum. 2, 1996, pp. 26-29 (Review)

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