Vigdis Lauvrak Bio 4600- H2003 Phage display.

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Presentation transcript:

Vigdis Lauvrak Bio 4600- H2003 Phage display

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.

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

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

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 100 000 depending on method and nature of variant.

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

E.Coli F pilus ssDNA dsDNA (RF)

Gene 3 Gene 8

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 3 phage 33 phage 8* phage 88 Selective infective phage/phagemid phagemid 3 phagemid 3+ phagemid 8* phagemid 8+ *Only small peptides tolerated + Additional wildtype protein supplied by helper phage.

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

Affinity selection

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

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

Filamentous phage antibody libraries ANTIGEN BINDING ScFv Fab ANTIGEN BINDING

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

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.

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).

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, 1,4 Benzodiazepine Libraries...…