Transcript Surface Display

Surface Display
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Phage
Bacteria
Yeast
Ribosome
Displayed: passenger protein
Anchoring motif: carrier protein
Phage Display
Panning
Filamentous phages are flexible rods about 1 mm long and 6 nm in
diameter, composed mainly (87% by mass) of a tube of helically
arranged molecules of the 50-residue major coat protein pVIII22; there
are 2700 copies in wild-type virions, encoded by a single phage gene VIII.
Inside this tube lies the single-stranded viral DNA (ssDNA; 6407-8
nucleotides in wild-type strains). At one tip of the particle there are five
copies each of the minor coat proteins pIII and pVI (genes III and VI,
respectively); minor coat proteins pVII and pIX (genes VII and IX) are at
the other tip.
Three rounds of panning against
streptavidin at constant stringency (0.5% Tween)
His-Pro-Gln (HPQ) as a consensus binding motif
specific for the
biotin binding site of streptavidin
Epitope mapping of the anti-FLAG M2
monoclonal antibody.
In order to determine which elements of the reported
FLAG epitope sequence Asp-Tyr-Lys-Asp-Asp-Asp-AspLys (DYKDDDDK) are required for antibody binding
Capture of the antibody by Protein A-agrose
• Protein A is a cell wall protein deriving
from Staphylococcus aureus which
exhibits unique binding properties for IgG
from a variety of mammalian species and
for some IgM and IgA as well. It binds with
the Fc region of immunoglobulins through
interaction with the heavy chain.
Epitope mapping of an anti-β-endorphin monoclonal
antibody with the Ph.D.-12 library
Cell surface display
Gram (-)
cell
Ice nucleation factor
promoting ice forming
Ice Nucleation
Protein (INP)
OmpC
Gram(+) cell
Staphylococall
Protein A
Yeast Surface Display
Screening Yeast Displayed Libraries
Phage vs Ribosome Display
Phage
display :(purple)
ribosome display:
(green)
Ribosome display
The DNA library constructs contain all the signals
required for cell-free in vitro transcription and translation.
The absence of a stop codon at the end of the coding
sequence prevents the release of the mRNA and the
nascent polypeptide from the ribosomes.
Low temperatures and an elevated level of magnesium
ions further stabilize the ternary mRNA–ribosome–
polypeptide complex. An unstructured tether is added to
the C-terminus of the constructs to enable correct folding
of the nascent polypeptides outside the ribosome tunnel.
After selection, mRNA molecules are recovered by
reverse transcription (RT) and amplified by polymerase
chain reaction (PCR).