Therapeutic Proteins

Question 1

Why do we want to express and purify proteins?

Answer 1

Therapeutics - Using biological systems we can produce a class of new drugs called biologics. These are a much larger class of drugs, and include examples such as infliximab and ocrelizumab. In 2017, the biologics market was valued at 17 billion and 12 were approved by the FDA, and 10 by the EMA. The most common therapeutic area is cancer, while AD has the least biologics. 
Basic scientific applications - RNA pol/restriction enzymes etc.

Question 2

What are the three ways of producing a protein? Highlight some adv/disadv for each.

Answer 2

Natural Source - No chemistry/DNA manipulation, cofactors already assembled, folded properly, only good for high abundance proteins
De Novo - No DNA manipulation, pure, established systems. Expensive and only good for proteins less than 50 AA
Expression system - established systems, high yield, reproducible. Cloning required and troubleshooting

Question 3

What are the two enzymes involved in DNA cloning?

Answer 3

EcoRI and DNA Ligase

Question 4

What is a plasmid?

Answer 4

A circular piece of DNA that replicates independently of host chromosomal DNA. Mainly found in bacteria. Confer antibiotic resistance, and contribute to virulence

Question 5

What are the key features of an expression vector?

Answer 5

Origin of replication
Promoter
Selectable marker
Multiple cloning site
Purification tag

Question 6

What are the different types of expression systems?

Answer 6

Bacteria (T7 late promoter and T7 RNA polymerase IPTG inducible)
Yeast (YIP, YRP, YEP)
Insect (Linearised baculovirus vector, polyhedron promotor)
Mammalian Cells (Cytomegalovirus promoter, TET operator, Tetracycline inducible)

Question 7

What are the key questions when selecting an expression system?

Answer 7

1. Type of protein
2. Solubility
3. PTMs
4. Codon Usage
5. Toxictiy

Question 8

What is a plantibody?

Answer 8

A plantibody is an antibody that is produced by plants that have been genetically engineered with animal DNA encoding a specific human antibody known to neutralize a particular pathogen or toxin. They are not yet approved for human use, however trials are being conducted on Ebola vaccine, HIV vaccine and Malaria vaccine.