L11 Role of phage display in antibody production

Question 1

What is phage display?

Answer 1

An in vitro selection technique using peptide genetically fused to the coat protein of a bacteriophage.

Question 2

What is a bacteriophage?

Answer 2

A virus that infects bacteria.

Question 3

How does a bacteriophage infect bacterial cells?

Answer 3

Inject their genetic material into cell, which is carried enclosed in outer protein shell known as ‘caspid’

Question 4

Where are fusion proteins added?

Answer 4

Mostly fused to protein p3.

p8 can also be used.

Question 5

How is GOI added to phage?

Answer 5

Using a phagemid.

construct for expression of single chain variable F chain (SvFv) as fusion protein with p3 gene.

Question 6

How does ScFv differ from the Fab?

Answer 6

Very similar. still binds to antigen, but heavy and light chain are linked by peptide linker, and only one subunit of VH and VL used.

Question 7

Which antibody fragments are typically expressed?

Answer 7

Fab, scFc or FV.

Question 8

What effect does length of linker protein in scFv’s have in fragments to function?

Answer 8

If too short, fragments dimerise and form ‘diabodies’

diabodies much higher affinity to their target.

Question 9

How can the use of diabodies differ to typical Ab fragments?

Answer 9

Bind much stronger to target, can be used in therapy and much lower doses needed. Highly specific in targeting of tumours in vivo.

Question 10

If shorter linkers are used, what else can be formed?

Answer 10

Triabodies and tetrabodies, even higher affinity.

Question 11

What happens if scFvs are mixed?

Answer 11

Bi-specific abs can be produced in this way.

Bivalt and trivant, targetting multiple antigens.

Question 12

Which phages are commonly used?

Answer 12

m13 (f1) and FD (T4)

Question 13

What steps are involved in phage display?

Answer 13

1) Creation of vector
2) Transformation of bacteria
3) Binding/Selection
4) Wash
5) Elution
6) Amplification

Question 14

What two features of a plasmid are combined with phage to make phagemid vector

Answer 14

Antibitic resistance and ORI.

M13 ORI

Question 15

What is phagemid added to to replicate?

Answer 15

Typically doesn’t encode any viral structural or replication proteins. Need helper phage to replicate.

Question 16

What is the procedure for phage display?

Answer 16

PCR amp of Ig VH and VL genes.

Cloning of VH and VL genes into phage(mid) vector, and expression a a fusion with a phage protein.

Identification of recombinant phage expressing the Ab by affinity selection (with antigen)

Production of solulbe form of Ab in E. coli

Question 17

How is scFv gene generated?

Answer 17

Reverse transcription of light and heavy chain mRNA from hybridoma, PCR to amplyify, then attached by linker.

Question 18

What is panning and how does it allow for selection of best bacteriophage?

Once selected, how is pure antibody obtained?

Answer 18

Using a column with immobilised antigen, the library of phages are washed over, ‘panned’

The bacteriophage with fusion proteins complementary to antigen are immobilised, the rest elute off.

Using column & magnetic beads, able to isolate this phage.

Once isolated, DNA isolated, and added to non-suppressor strain. Non-suppressor strain expressed antibody fragment, which can then be isolated.

Question 19

What is the role of the amber stop codon?

Answer 19

It is a switch between displayed antibody fragment and soluble form.

In suppressor bacterial strain, stop codon is not ‘read’ and whole protein synthesised.

In non-suppressor strain, stop codon read and soluble scFc produced.

Question 20

In non-suppressor strain infected e.coli, how is antibody obtained?

Answer 20

Soluble Ab may be present in culture supernatant (secreted by bacterial cell), in the bacterial periplasm and/or inside the bacterial cytoplasm.

Different means of purifying for each, but generally affinity purification using antigen.

Question 21