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Biotechnology > Conjugation and cross-linking methods > Flashcards

Conjugation and cross-linking methods Flashcards

1
Q

What are some biological tools uses for conjugation?

A

Affinity columns
Probes
Diagnostic

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2
Q

How does conjugatioin improve therapeutics?

A

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

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3
Q

What are diagnostic biosensors?

A

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

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4
Q

What is palmitoylation?

A

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

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5
Q

Name PTMs.

A 
Glycoyslation
Palmitolyation
Acetylation
Phohsphorylation
Disulphide bridges
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6
Q

What is required for a PTM?

A

A consensus sequence

Accessibility to an enzyme

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7
Q

What allows for the addition of non-natural groups?

A

Chemical/synthetic conjugation

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8
Q

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

A

No

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9
Q

How many mutation are required to introduce a chemical conjugation ?

A

1

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

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10
Q

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

A

Lysine

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11
Q

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

A

Cysteine

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12
Q

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

A

Serine

Threonine

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13
Q

What is a big advantage of synthetic conjugation?

A

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

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14
Q

What is a disadvantage of synthetic conjugation?

A

Can affect structure/function

Limited to non-damaging chemistry

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15
Q

How is conjugation used in affinity columns?

A

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

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16
Q

What are used probes for?

A

To detect the presence or localization of something

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17
Q

What types of probes exist?

A

Radioactive
Fluorescent
Chemiluminsecent
Enzymes

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18
Q

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

A 
Narrow emission
(so no overlap in range)
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19
Q

How does an ELSIA sandwich work?

A

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

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20
Q

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?

A

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

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21
Q

Alternatively can the antigen be directly immobilised to the surface?

A

Yes

This would remove the need for the ‘first’ antibody

22
Q

What is FITC?

A

Fluorscein isothiocyanate -

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

23
Q

What is FITC use for?

A

Immunohistochemistry

24
Q

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

A

It still has one variable region hence still able to bind

25
Q

What is the function of glutaraldehyde in cross-linking?

A

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

26
Q

What group does glutaraldehyde react with?

A

Free amine groupss

27
Q

How do photoreactive crosslinkers help study protein interactions?

A

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)

28
Q

Describe a photoreactive crosslinker.

A

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

29
Q

In a photoreactive crosslinker what group in photoreactive?

A

The triple N

very reactive and will bind almost anything

30
Q

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

A

Fluorescence doesn’t penetrate tissue

31
Q

Why can radioactive probes also be used as a therapy?

A

To cause DNA damage to tumour cells

32
Q

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

A

Iodine 131 can be attached to a tyrosine

e.g. Iodine-131 tositumomab - anti-CD20 radio-immunoconjugation therapy for non-hodgkin lymphoma

33
Q

How to chelators work?

A

Absorb metal from substances

These metals can be radioactive and chelators can be conjugated as a tag

34
Q

What is a ADC?

A

Antibody drug conjugate

- conjugate a drug to antibody

35
Q

What is photodynamic therapy?

A

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

36
Q

What is ADEPT?

A

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

37
Q

What is a bi-specific antibody?

A

Reduce antibodies to two half and recombine two different halves

38
Q

Give an example of a bi-specific antibody?

A

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

39
Q

What is PEG?

A

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

40
Q

Is PEG biodegradable?

A

No

Hence can accumulate I liver and may become not inert

41
Q

What is the function of PEG?

A

Increase stability and decrease clearance.

42
Q

What is an issue with PEG?

A

Not biodegradable hence accumulates in liver

43
Q

What is click chemistry?

A

Modularised groups can that be conjugated together

44
Q

Is click chemistry stereospecific?

A

Yes

45
Q

What is the theoretical yield of click chemistry?

A

~100%

46
Q

Name a common form of click chemistry?

A

The Huisgen azide-alkyne cycloaddition

47
Q

What is biotinylation?

A

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

48
Q

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

A

TAG - stop codon

49
Q

How do you create artificially charged tRNAs?

A

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

50
Q

What happen if the tRNA is not charged?

A

The protein gets truncated

Biotechnology (13 decks)

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