Conjugation and cross-linking methods

Question 1

What are some biological tools uses for conjugation?

Answer 1

Affinity columns
Probes
Diagnostic

Question 2

How does conjugatioin improve therapeutics?

Answer 2

Better stability
Low immunogenecity (cover epitopes)
Reuced clearance
Drug delivery

Question 3

What are diagnostic biosensors?

Answer 3

Biosensors combine a transducer with a biorecognition element and thus are able to transform a biochemical event on the transducer surface directly into a measurable signal

Question 4

What is palmitoylation?

Answer 4

The covalent attachment of fatty acids, such as palmitic acid, to cysteine and (sometimes serine and threonine )
Typically to TM proteins

Question 5

Name PTMs.

Answer 5

Glycoyslation
Palmitolyation
Acetylation
Phohsphorylation
Disulphide bridges

Question 6

What is required for a PTM?

Answer 6

A consensus sequence

Accessibility to an enzyme

Question 7

What allows for the addition of non-natural groups?

Answer 7

Chemical/synthetic conjugation

Question 8

Is chemical conjugation restricted by structural motif or enzyme recognition sites?

Answer 8

No

Question 9

How many mutation are required to introduce a chemical conjugation ?

Answer 9

1

E.g. introduce ASN and not the whole consensus sequence

Question 10

What base would you engineer to introduce for amine based linkages?

Answer 10

Lysine

Question 11

What base would you engineer to introduce a for thiol based linkage?

Answer 11

Cysteine

Question 12

What base would you engineer to introduce for hydroxyl based linkage?

Answer 12

Serine

Threonine

Question 13

What is a big advantage of synthetic conjugation?

Answer 13

High specificity
Also accepted answers:
High yield
Can harness naturally occurring aa

Question 14

What is a disadvantage of synthetic conjugation?

Answer 14

Can affect structure/function

Limited to non-damaging chemistry

Question 15

How is conjugation used in affinity columns?

Answer 15

Conjugate POI to solid surface by a covalent link (e.g. protein A to an agarose bead)

Question 16

What are used probes for?

Answer 16

To detect the presence or localization of something

Question 17

What types of probes exist?

Answer 17

Radioactive
Fluorescent
Chemiluminsecent
Enzymes

Question 18

Chemical fluorophores are designed to have very ………… emission s they can be distinguished between.

Answer 18

Narrow emission
(so no overlap in range)

Question 19

How does an ELSIA sandwich work?

Answer 19

Attach antibody to plate
Wash over solution - let antigen bind
Add second antibody
Protein sandwiched between two antibodies
Second antibody can be conjugated to fluorophore or a third antibody probe can be used to tag the second antibody

Question 20

Why in the ELISA sandwich assay is the use of a third conjugated antibody preferred over the use of a fluorophore/enzyme conjugated second antibody?

Answer 20

The second antibody needs to be specific to the antigen whereas the third antibody only needs to be specific to a constant region of an antibody. Hence the same third antibody can be used for different antigen hence more efficient to use a single antigen

Question 21

Alternatively can the antigen be directly immobilised to the surface?

Answer 21

Yes

This would remove the need for the ‘first’ antibody

Question 22

What is FITC?

Answer 22

Fluorscein isothiocyanate -

Is a fluorescent conjugate that binds to amine groups via its isothiocynate group

Question 23

What is FITC use for?

Answer 23

Immunohistochemistry

Question 24

How does a half antibody conjugated to a protein via its cysteine bind its antigen?

Answer 24

It still has one variable region hence still able to bind

Question 25

What is the function of glutaraldehyde in cross-linking?

Answer 25

Cross links protein in close proximity It is also used to fix cells

Question 26

What group does glutaraldehyde react with?

Answer 26

Free amine groupss

Question 27

How do photoreactive crosslinkers help study protein interactions?

Answer 27

Photoreactive crosslinkers links complexes (binding partners) during exposure to UV light Linked complexes can then be purified and the ligand idenitified (by mass spec or peptide sequencing)

Question 28

Describe a photoreactive crosslinker.

Answer 28

Hetrerobifunctional linkers with amine reactive NHS easter on one end (to bait known protein) and a photoreactive group on the other

Question 29

In a photoreactive crosslinker what group in photoreactive?

Answer 29

The triple N | very reactive and will bind almost anything

Question 30

Why would you use radioactive probes over fluorescent probes in whole body imaging?

Answer 30

Fluorescence doesn't penetrate tissue

Question 31

Why can radioactive probes also be used as a therapy?

Answer 31

To cause DNA damage to tumour cells

Question 32

Give an example of a radio active probe being used as a therapy.

Answer 32

Iodine 131 can be attached to a tyrosine | e.g. Iodine-131 tositumomab - anti-CD20 radio-immunoconjugation therapy for non-hodgkin lymphoma

Question 33

How to chelators work?

Answer 33

Absorb metal from substances | These metals can be radioactive and chelators can be conjugated as a tag

Question 34

What is a ADC?

Answer 34

Antibody drug conjugate | - conjugate a drug to antibody

Question 35

What is photodynamic therapy?

Answer 35

Uses the photoreactive species to deliver toxic free radical to target cell to induce cell death

Question 36

What is ADEPT?

Answer 36

Antibody enzyme prodrug therapy. Prodrug is in circulation Enzyme to activate drug localised by antibody to specific cell type

Question 37

What is a bi-specific antibody?

Answer 37

Reduce antibodies to two half and recombine two different halves

Question 38

Give an example of a bi-specific antibody?

Answer 38

Removab Designed to bring tumour cells in contact with T-cells (recognises CD3 and epCAM)

Question 39

What is PEG?

Answer 39

A polyethylene glycol which increases the hydrodynamic volume leading to decreased clearance

Question 40

Is PEG biodegradable?

Answer 40

No | Hence can accumulate I liver and may become not inert

Question 41

What is the function of PEG?

Answer 41

Increase stability and decrease clearance.

Question 42

What is an issue with PEG?

Answer 42

Not biodegradable hence accumulates in liver

Question 43

What is click chemistry?

Answer 43

Modularised groups can that be conjugated together

Question 44

Is click chemistry stereospecific?

Answer 44

Yes

Question 45

What is the theoretical yield of click chemistry?

Answer 45

~100%

Question 46

Name a common form of click chemistry?

Answer 46

The Huisgen azide-alkyne cycloaddition

Question 47

What is biotinylation?

Answer 47

Attaching biotin to antibody to take advantage of its affinity for streptavidin that you can conjugate to a POI

Question 48

In unnatural amino acid addition which codon is recognised as the new codon?

Answer 48

TAG - stop codon

Question 49

How do you create artificially charged tRNAs?

Answer 49

Engineer biorthogonal tRNA synthetase and tRNA pair to recognise a non-natural amino acid

Question 50

What happen if the tRNA is not charged?

Answer 50

The protein gets truncated