Conjugation & Cross-linking

Question 1

What is conjugation?

Answer 1

The modification of proteins to attach an additional group such as chemical prosthetic groups, fusion proteins, PTMs.

Question 2

Why should you conjugate?

Answer 2

Improve the stability
Enhance the function
Label a protein

Question 3

Give examples of where conjugation is useful.

Answer 3

Affinity columns
Antibody fluorescent probes
Biosensors
Therapeutic proteins
Drug delivery

Question 4

Describe some natural conjugation methods/ PTMs.

Answer 4

PTMs are natural ways to alter proteins by function, stability or other properties. They are site specific and have temporal and localised control.

• Glycosylation - stability, folding, drug clearance; Asn, Ser/Thr
• Palmitoylation - protein-protein interactions, localisation; Cys, Ser/Thr
• Acylation - stability, degredation signals; Lys, N-terminus
• Phosphorylation - regulates activity; Ser, Thr, Tyr, His
• Disulphide bridges - stability; Cys

Question 5

Why are chemical modifications useful?

Answer 5

Chemical modifications allow you to control or enhance properties. There’s no need for specific recognition sites which can be advantageous. Conjugations can affect function, activity and stability and in many cases the sites already exist. These chemical reactions need to occur in cellular conditions but they are specific and are high yielding.

Question 6

What side chains are exploited?

Answer 6

Exploited side chains include: lysine, cysteine, serine, threonine, tyrosine, N-terminus and C-terminus.

Question 7

Describe the 7 tools that are applications of conjugation

Answer 7

#Affinity columns - used in purification to immobilise the target e.g. proteinA and agarose beads
#Probes - used in localisation and labelling so can be measured using microscopes, flow cytometry, fluorimetry e.g. enzymes, radioisotopes, fluorophores
#Fluorescent probes - Allows fluorescent tracking. e.g. FITC, Fluroscien-5-maleimide
#Enzyme-antibody conjugates - used in ELISA or western blots to measure activity e.g. horse radish peroxidase or alkaline phosphatase
#Glutaraldehyde cross-linking - found in protein-protein interactions and used to fix samples e.g. reacts with amine gorups on enzymes and antibodies
#Photoreactive crosslinkers - This uses a bait and trap protein and small molecule, that is activated by UV so proteins in close proximity bind.
#Radiolabelled proteins- used in imaging or therapeutic delivery, can also be chelators that absorb metals from solutions.

Question 8

Describe 5 therapeutic applications of conjugation

Answer 8

#Antibody-Drug Conjugates - This connects small drugs to antibodies and is often used as cancer treatments. e.g. anti CD33 drug-linked antibody + calicheamicin 
#Photodynamic Therapy - photoreactive species deliver toxic free radicals to induce cell death. This is localised action
#Antibody-Enzyme Pro-drug Therapy (ADEPT) - This is made recombinantly. The antibody targets the cell and the enzyme converts metabolites to active substances.
#Bi-specific Antibodies - Two antibodies fused together that can bind 2 different cells or proteins. e.g. Removab; recognises CD3 and EpCAM.   
#Modulating Pharmacokinetics - Increase the time the drug or protein stays in the body by adding polymers to mask certain aspects of the protein. Some processes you can do include: PEGylation, dextran, polysiaylation, HAPylation, PASylation, methyalacrylamide, hydroxy-ethyl starch.

Question 9

Describe click chemistry

Answer 9

Click chemistry allows modular attachments of chemical building blocks with high yield and specificity under gentle conditions. Protein engineering is required as the chemistry occurs between an alkyne and a azide, so the protein needs to have one of these groups.

Question 10

Describe biotinylation

Answer 10

Biotin is a molecule that acts like a ligand to avidin/ streptavidin. This is useful in biotech applications such as resins.

Question 11

Describe non-natural amino acids

Answer 11

The aim is to engineer a stop codon, tRNA and tRNA synthetase so that a non-natural amino acids can be carried, added ad recognised in the translation process.
Stop codon is often TAG, delete release factor 1; normally engineered from Mj to use in E.coli.
e.g. p-AcF can form oximes
p-AzF can do click chemistry

Question 12

What are the considerations for biotech applications?

Answer 12

• if an improvement occurred
• expression systems
• efficacy
• easy
• cheap
• reproducibility
• homogeneity
• solubility
• purification systems
• high- throughput

Question 13

What are the considerations for pharmaceutical applications?

Answer 13

• efficacy
• target penetration
• reproducibility
• homogeneity
• drug leaching
• pharmacokinetics
• clearance
• degredation
• drug loading capabilties
• specificity
• toxicity
• delivery system
• expression systems
• immunogenecity