Conjugation and cross-linking methods Flashcards
What are some biological tools uses for conjugation?
Affinity columns
Probes
Diagnostic
How does conjugatioin improve therapeutics?
Better stability
Low immunogenecity (cover epitopes)
Reuced clearance
Drug delivery
What are diagnostic biosensors?
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
What is palmitoylation?
The covalent attachment of fatty acids, such as palmitic acid, to cysteine and (sometimes serine and threonine )
Typically to TM proteins
Name PTMs.
Glycoyslation Palmitolyation Acetylation Phohsphorylation Disulphide bridges
What is required for a PTM?
A consensus sequence
Accessibility to an enzyme
What allows for the addition of non-natural groups?
Chemical/synthetic conjugation
Is chemical conjugation restricted by structural motif or enzyme recognition sites?
No
How many mutation are required to introduce a chemical conjugation ?
1
E.g. introduce ASN and not the whole consensus sequence
What base would you engineer to introduce for amine based linkages?
Lysine
What base would you engineer to introduce a for thiol based linkage?
Cysteine
What base would you engineer to introduce for hydroxyl based linkage?
Serine
Threonine
What is a big advantage of synthetic conjugation?
High specificity
Also accepted answers:
High yield
Can harness naturally occurring aa
What is a disadvantage of synthetic conjugation?
Can affect structure/function
Limited to non-damaging chemistry
How is conjugation used in affinity columns?
Conjugate POI to solid surface by a covalent link (e.g. protein A to an agarose bead)
What are used probes for?
To detect the presence or localization of something
What types of probes exist?
Radioactive
Fluorescent
Chemiluminsecent
Enzymes
Chemical fluorophores are designed to have very ………… emission s they can be distinguished between.
Narrow emission (so no overlap in range)
How does an ELSIA sandwich work?
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
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?
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
Alternatively can the antigen be directly immobilised to the surface?
Yes
This would remove the need for the ‘first’ antibody
What is FITC?
Fluorscein isothiocyanate -
Is a fluorescent conjugate that binds to amine groups via its isothiocynate group
What is FITC use for?
Immunohistochemistry
How does a half antibody conjugated to a protein via its cysteine bind its antigen?
It still has one variable region hence still able to bind
What is the function of glutaraldehyde in cross-linking?
Cross links protein in close proximity It is also used to fix cells
What group does glutaraldehyde react with?
Free amine groupss
How do photoreactive crosslinkers help study protein interactions?
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)
Describe a photoreactive crosslinker.
Hetrerobifunctional linkers with amine reactive NHS easter on one end (to bait known protein) and a photoreactive group on the other
In a photoreactive crosslinker what group in photoreactive?
The triple N
very reactive and will bind almost anything
Why would you use radioactive probes over fluorescent probes in whole body imaging?
Fluorescence doesn’t penetrate tissue
Why can radioactive probes also be used as a therapy?
To cause DNA damage to tumour cells
Give an example of a radio active probe being used as a therapy.
Iodine 131 can be attached to a tyrosine
e.g. Iodine-131 tositumomab - anti-CD20 radio-immunoconjugation therapy for non-hodgkin lymphoma
How to chelators work?
Absorb metal from substances
These metals can be radioactive and chelators can be conjugated as a tag
What is a ADC?
Antibody drug conjugate
- conjugate a drug to antibody
What is photodynamic therapy?
Uses the photoreactive species to deliver toxic free radical to target cell to induce cell death
What is ADEPT?
Antibody enzyme prodrug therapy.
Prodrug is in circulation
Enzyme to activate drug localised by antibody to specific cell type
What is a bi-specific antibody?
Reduce antibodies to two half and recombine two different halves
Give an example of a bi-specific antibody?
Removab
Designed to bring tumour cells in contact with T-cells
(recognises CD3 and epCAM)
What is PEG?
A polyethylene glycol which increases the hydrodynamic volume leading to decreased clearance
Is PEG biodegradable?
No
Hence can accumulate I liver and may become not inert
What is the function of PEG?
Increase stability and decrease clearance.
What is an issue with PEG?
Not biodegradable hence accumulates in liver
What is click chemistry?
Modularised groups can that be conjugated together
Is click chemistry stereospecific?
Yes
What is the theoretical yield of click chemistry?
~100%
Name a common form of click chemistry?
The Huisgen azide-alkyne cycloaddition
What is biotinylation?
Attaching biotin to antibody to take advantage of its affinity for streptavidin that you can conjugate to a POI
In unnatural amino acid addition which codon is recognised as the new codon?
TAG - stop codon
How do you create artificially charged tRNAs?
Engineer biorthogonal tRNA synthetase and tRNA pair to recognise a non-natural amino acid
What happen if the tRNA is not charged?
The protein gets truncated
