Lecture 9

Question 1

Define: Recombinant proteins

Answer 1

​Proteins derived from the expression of recombinant DNA within living cells

Question 2

Requirements of a bacterial expression system?

Answer 2

1. Expression vector (plasmid)

2. Host bacterial cells (often modified)

Question 3

Requirements for HIGH LEVEL expression:

Answer 3

1. A strong regulated promoter (vector): make large numbers of mRNA transcripts, able to produce when required
2. Effective translation of proteins from mRNA transcripts
   (host cells)
3. Correct processing and folding of the translated proteins
   (host cells)

Question 4

Features of an E.coli expression vector

Answer 4

Promoter
Operator 
Polylinker
Selectable genetic marker 
Origin of replication
Gene encoding repressor that binds O and regulates P

Question 5

Steps in producing recombinant proteins?

Answer 5

1. Insert gene (encodes protein to be expressed) into bacterial expression vector
   a- digest vector and insert with REs
   b- ligate gene insert into bacterial expression vector (at MCS) using DNA ligase
2. Transform recombinant expression vector into host cells (E.coli)
3. Select for transformed E.coli host cells using selectable antibiotic marker (DNA sequencing to ensure correct clone)
4. Express protein using inducible promoter
5. Purify protein