Phage display Flashcards
Phage display
a technique in which antibody fragments are expressed as a fusion coat protein on bacteriophage
fused proteins (antibody fragments)
are displayed on the surface of the vision, while the DNA encoding the fusion resided within the virion
DNA encoding the antibody (fusion)
is found within the virion
what sort of phage is used
a filamentous phage structyre
name an example of a filamentous phage
M13 phage
how long in length
200nm
what sort of genome
ssDNA
target antibody to be expressed
on the outside of the vision
process of making a phage display library
1) B cell source (immunised animal)
2) production of mRNA
3) production of cDNA from mRNA
4) PCR to amplify each the the V-gene facilities ( VH and VL)
5) clone as scFv onto PIII of phage
scFv =
Vl and Vh of an immunogoblin linked by a peptide spacer
what allow expression of scFv on the surface of phages
fusion proteins between scFV and PIII minor coat proteins allow expression
where are PIII minor coat proteins found
on the end of the phage
Fab fragments of antibodies can be expressed on the
surface of a phage as fusion proteins with PIII
Fab fragments of antibodies can also be expressed on the surface of phages as fusion proteins with
pVIII
where is pVIII expressed
along the length of the phage particle
pVIII are useful for
the isolation of low affinity antibodies
phage selection
1) phage antibody library
2) biopanning and washing
3) UV source and shadow stick selection
4) elution of bound phage antibodies
5) add to agar plate with e.coli and infection
various different places where antibodies can be expressed on a phage
- mainly expressed on the end (PIII) or along the length (PVIII)
end goal of phage display
is to identify a phage producing an antibody with complementary VH and VL fragments of interest
isolating phages
take population of phages- do ELISA and this allows you to pull out the phage producing the antibody of interest
phage selection
need to eliminate all phages not producing antibody of interest
- UV used to get rid of phages not of interest
- phage of interest are protecting
- these phages are then eluted and used to infect e.coli
phage display process overview
1) take genes that encode a specific antibody and combine them on a strand of DNA
2) then the DNA is packaged into a protein coat made from a virus which only infects bacteria (bacteriophage)
3) when antibody gene are transcribed and antibody hat is formed not he top of the phage
4) phage antibody ‘hats’ are unique and bind to a specific disease target molecule 9change the antibody gene and you change the type of antibody hat)
5) many antibody phage with different hats are produced
6) library is a source of dining new antibody medicine
to target particular disease molecules
the correct phage antibody has to be selected from the library
e.g. pathogen/antigen can be was he over the library to see which antibody complexes with it
once a phage displaying the desired antibody has be identified
take phage and infect bacteria e.g. e.coli
