1. Vigdis Lauvrak Bio H2003
Phage display

2. DNA can be directly amplified by replication or PCR
DNA can be directly amplified by replication or PCR. Proteins can not be directly amplified.
Phage display is based on the display of a peptide or protein on a phage surface and a gene encoding it inside the phage particle.

3. Coupling genotype to the phenotype
DNA
DNA
DNA
DNA
DNA
DNA
DNA
DNA
DNA
Selection from large samples

4. Evolution in the test tube.
DNA
DNA
DNA
DNA
DNA
DNA
An increased number of selectable variants increased affinity increased specificity or increased activity.

5. A distinction is made between selection and screening.
Screening: Individual variants (or pools of variants) are placed in wells, on grids, pins, chips etc.
In selection interesting variants are pulled out from pools of variants based on property.
The chenical structure may be identified by a given position, by direct sequencing (peptide) or sequencing after amplification (DNA/RNA/ amplification of variant producing cell). In addition there are systems for tagging individual variants with a code that can somehow be recognised. There is a limitation on the number of variants that can be screened ranging from a few hundred to depending on method and nature of variant.

6. Filamentous phages (M13, fd, f1).
pIII
pIII
pVI
pVIII
gpVII
gpIX

7. E.Coli F pilus
ssDNA
dsDNA (RF)

8. Gene 3
Gene 8

9. phage 3 phage 33 phage 8* phage 88
-Displayed insert
-pIII
-Truncated pIII
-gene3
-gene8
-Inserted DNA
-Inducible promoter
-Target fused to N- terminal pIII fragment
phage phage phage 8* phage 88
Selective infective phage/phagemid
phagemid phagemid phagemid 8* phagemid 8+
*Only small peptides tolerated
+ Additional wildtype protein supplied by helper phage.

10. Replication- origon Helper phage Plasmid DNA with f1-ori , and
RF-DNA
Encodes 10 phage proteins, replication origon and an intergenic region
Helper phage
Plasmid DNA with
f1-ori , and
E.coli ori .
Phage proteins

11. Affinity selection

13. X10GAA……………....gpIII X3LLGGX3GAA…......gpIII CX3LLGGX3 CGAA…gpIII
FILAMENTOUS PHAGE RANDOM PEPTIDE LIBRARIES
(NNK)
(NNM)
Synthetic oligo nucleotides with random regions are created and inserted into the gene encoding a phage coat protein
X10GAA……………....gpIII
X3LLGGX3GAA…......gpIII
CX3LLGGX3 CGAA…gpIII
CX6CGAA……………gpIII
CX9CGAA……………gpIII
Peptides with a random amino acid sequence are displayed on the phage surface

14. NNK NNS Any amino acid and a reduced number of stop codons (only TAG).
Nucleotide symbol table
Symbol Specification
A Adenine
C Cytocine
G Guanine
I Inosine
T Thymine
U Uracil
M A + C
R A + G
W A + T
S C + G
Y C + T
K G + T
V A + C + G
H A + C + T
D A + G + T
B C + G + T
N A + C + G + T
O 5Me-dC
Q 5Me-dU
Any amino acid and a reduced number of stop codons (only TAG).
NNK
NNS

15. Filamentous phage antibody libraries
ANTIGEN
BINDING
ScFv
Fab
ANTIGEN
BINDING

17. Fab-library:
Periplasma VL
VH-gpIII fusion
P Leader VH CH Leader VL CL

18. Bacteriophages as genetic packages:
Small genomes,
Few proteins,
Easely amplifiable,
Many copies per unit volume (compared to bacteria and yeast),
Inserts are tolerated in many virus coat proteins
Filamentous phages are highly stable at a number of normally denaturating conditions like high or low pH and extreme salt.

19. Phage display:
Selection of small molecular weight mimetics of larger molecules molecules (peptide display).
Selection of peptides and proteins with novel functions and specificities (antibody display).
Directed Molecular evolution
Selection of proteins with novel specificities
Improvement of affinity
Improvement of stability
improvement of enzymatic activity
Why?
To make superior therapeutic and diagnostic molecules.
To make molecules with new properties (of industrial value).
To increase knowledge (How can nature solve a particular problem).

20. Combinatorial libraries:
Large samples of variants were the building blocks (amino acids, bases, sugars and other molecules) are combined in a (more or less) random fashion.
Phage display libraries,
Protein/Peptide display on bacteria,
Protein/Peptide display on yeast,
Peptides on plasmids,
Ribosomal display
Synthetic peptide libraries,
Nucleic acid libraries,
N-substituted Glycine Peptoid libraries,
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