I am working with phage displayed library construction. Our lab constructed pIII-displayed peptide (CX6C, CX9C, CX12C) library using M13KE (Ph. D. Cloning system NEB #E8101). Instead of randomized peptide, I am working on display disulfide bond constrained mini protein library on M13 phage. I have a mini protein template contained 60 amino acids and 2 pair of disulfide bond and I want to display this mini-protein with mutations on 4 sites on M13 phage.
According to the NEB phage display manual, the inserted pieces longer than 30-50 amino acids may have a deleterious effect on the infectivity function of pIII.
Is there any thing I can do to alleviate this deleterious effect OR is there any other phage platform that better fit into my project?
Phage display of antibody scFv fragments (ca. 200 amino acids, 2 disulfide bridges) is no problem. https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0307.pdf
scFv can be expressed at the N-terminus of pIII , or fusions to the C-terminal domain of pIII can be co-expressed with wt gIII provided by a helper phage. The second option (monovalent display) is preferable when selecting for high affinity
https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0134.pdf
However, we have found it useful to stringently control expression of gIII-fusions, as background expression of toxic fusion proteins can lead to elimination of such phasmids:
"Expression of toxic gene products affects bacterial cell growth and phage display, causing a strong selection against plasmid maintenance and integrity. During phage propagation steps, in particular, phagemid instability can dramatically affect diversity of antibody libraries or even lead to the deletion of antibody genes. We constructed a modified phage display vector by introducing a strong transcriptional terminator upstream of the lac promoter, which, together with glucose suppression of its CAP-dependent activation, very efficiently represses product formation before induction."
https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0107.pdf
A phage antibody library is created by cloning these repertoires as fusion proteins with a minor coat protein of bacteriophage (the gene 3 protein) (10–12). Each resulting phage has a functional antibody protein on its surface and contains the gene encoding the antibody incorporated into the phage genome.
Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-
In phage display technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to display the protein on the outside. ... In this way, large libraries of proteins can be screened and amplified in the process called in vitro selection, which is analogous to natural selection.
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