P1: Protein Expression Practical

Question 1

Benefits of E.coli expression system:

Answer 1

• Economical
• Rapid and easy to perform
• High level expression
• Several expression systems available
• Express as fusion proteins

Question 2

Disadvantages of E.coli as an expression system:

Answer 2

• Lacks machinery for protein modification
• Protein aggregation
• Need to optimise expression of mammalian proteins

Question 3

How are His tags purified?

Answer 3

• By immobilised metal affinity chromatography

Question 4

Outline some key factors that determine expression of cloned genes:

Answer 4

• Gene copy number
• Promoter activity
• mRNA stability
• Ribosome binding site
• Codon usage
• Protein stability
• Other factors (host-dependent)