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Wednesday, September 15, 2004

Bacteriophage display is on of my interests

I suspect thatit doesn't interest most of my few readers (so this is a blog).
In short it is a technique for finding antibodies that stick to a desired target which does not involve mice.

I think it is possible to use phage display to detect protein binding. The idea is to make two libraries of phage so that a phage from the different libraries have to stick together to grow.
A way to do this is to make two phages each of which has one essential gene with an amber nonsence mutation so that they complement each other. Mix infect e Coli with highly diluted phage mixture plate on non amber supressor e Coli then scrape and grow in amber suppressor e Coli.

I have wondered why phage display is not used to find T-cell receptors (TCR) which stick to a desired target. At the end of this long post I get around to saying that the two complementary libraries idea above might be useful to find T-Cell receptors specific for a desired target.

A search of Pubmed for "phage display TCR" returns 11 hits only two of which are about phage display of TCRs. The others use phage display to find antibodies that are like TCRs in that they are HLA restricted.

Search "Bacteriophage display TCR" returns the same two articles (this time with 28 articles on other topics)

Weidanz JA, Card KF, Edwards A, Perlstein E, Wong HC.
Display of functional alphabeta single-chain T-cell receptor molecules on the surface of bacteriophage. J Immunol Methods. 1998 Dec 1;221(1-2):59-76.

Onda T, LaFace D, Baier G, Brunner T, Honma N, Mikayama T, Altman A, Green

A phage display system for detection of T cell receptor-antigen interactions.
Mol Immunol. 1995 Dec;32(17-18):1387-97.
PMID: 8643108 [PubMed - indexed for MEDLINE]

Same for phage TCR
Same for phage display T Cell Receptor.

Onda et al hint as to the problem. The affinity of the TCR for its target is usually too low to be useful. They us a M-13 phage dsigned to present many copies of the TCR and still recognise that they got results only by luck.

"The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCR alpha chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCR alpha chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the alpha chains were derived. Cross-specificity analysis using two antigen-binding TCR alpha chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCR alpha-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding."

Now I had a thought that the TCRs could be made multivalent (but been done) . That is you want many TCRs on the phage near each other so when one drops the target the other grabs it.
A way to increase the number of TCRs stuck to each other is to stick bacteriophage together. This can be made to work as explained by Burge et al.

Burg M, Ravey EP, Gonzales M, Amburn E, Faix PH, Baird A, Larocca D.
Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery. DNA Cell Biol. 2004 Jul;23(7):457-62.

Still the key thing seems to be that killler TCR binds antigen well only when the antigen is presented in the cleft of HLA class I antigen. Since Onda et al others have developed well the title says it

Le Doussal J, Piqueras B, Dogan I, Debre P, Gorochov G.
Phage display of peptide/major histocompatibility complex.
J Immunol Methods. 2000 Jul 31;241(1-2):147-58.

I think the two libraries made with phage which have mutations in different essential genes might be a way use phage display of peptide/MHC complex to select phage displaying the desired TCR.

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