Overexpression of cloned genes

1
Overexpression of cloned genes can yield large
amounts of valuable proteins. Genes can be
overexpressed (1) in bacteria (e. g. E. coli
) (2) in eukaryotic cells
2
Many human proteins are used to treat
disease. These are normally present in the body
in trace amounts.
Protein Disease ?-antitrypsin
Emphysema Epidermal growth factor
Wound healing Insulin
Diabetes Erythropoietin Anaemia Factor
VII Haemophilia Factor IX
Haemophilia Growth hormone
Growth disorders Tissue plasminogen activator
Heart attacks
3
E. coli expression plasmids
Strong promoter (regulatable)
Unique restriction sites
e. g. Hin dIII
CATATG
Strong RBS
Nde I site
4
PCR is used to create an Nde I restriction site
at the start codon of the target gene
and another restriction site e. g. Hin dIII
downstream of the stop codon.
5
The amplified form of the gene (the PCR product)
has the restriction sites at each end.
Nde I
Hin dIII
6
CATATG
Nde I
Hin dIII
Inserting the amplified gene into the expression
vector places the start codon at the optimum
distance from the RBS (2 - 11 bp upstream).
7
The amplified gene must be re-sequenced to ensure
that there are no base changes. Many heat-stable
DNA polymerases have a high error rate. Taq
polymerase has no proof-reading exonuclease
activity and frequently mis-incorporates bases.
8
The pT7-7 system
pGP1-2
(KanR)
Inducible T7 RNA polymerase
Nde I HindIII
T7 promoter
(AmpR)
pT7-7 expression plasmid
9
T7 RNA pol - only recognises the T7 promoter (a
23 bp sequence) - initiates frequently -
transcribes DNA at 200bases/sec (4 x faster than
E. coli RNA pol)
10
Analysis of expression level by protein gel
electrophoresis.
11
Band of overproduced protein.
12
Target protein can be produced at a high level
up to 30 of total cellular protein Problems O
verproduced proteins may form inclusion
bodies, aggregates of incorrectly folded inactive
protein. Eukaryotic genes may have codon
usage which is unfavourable for expression in E.
coli. Specialised host strains have plasmids
with extra copies of genes for rare tRNAs.
13
Protein stability Recombinant proteins do not
accumulate if they are rapidly degraded by
proteases. The N-terminal AA affects stability.
N-terminal AA
Half-life Met, Ser, Ala, Thr, Val, Gly
20 hours Ile, Glu
gt 30 min Tyr, Gln
10 min Pro
7 min Phe, Leu, Asp, Lys
3 min Arg
2 min
Changing the first AA can improve stability.
14
Some expression vectors encode hexahistidine tags.
M H H H H H H M
RBS Start codon
Nde I
Tagged proteins bind specifically to immobilised
metal ion affinity columns. They can be purified
in one step.