Lecture notes on the topic Protein Expression and Engineering, taught by Professor Bernadette Byrne at Imperial College London. Course: Bsc Biochemistry. Module: Structural Biology.
Genetic elements involved in expression of plasmid-coded protein:
controls access
to promotor
by
inducing change
in DNA
polymerase binds,
MNA shape
drives gene expression ends transcription
codon
siteribosomalbindingonstartof gene the of
end of gene
of interest
gene drives plasmid replication to synthesise
sufficient amounts of protein
PROMOTERS
e.g. LacZ promoter I operator
region
↳
upstream Lac I
gene produce not accessible
synthesises Lac I protein
binds specifically to operator promoter region
:prevents polymerase
RNA
BINDING
from binding
a
used in bacteria binds, induces conformational
but not in labs so Lac I cannot bind to operator
Inot stable enough (
control and optimisation of expression
growth rate death rate,
=
no net ↑
Optimise by:
Optical 1.
Vary concentration of inducer
density 2. Vary temperature of growth
at600nm .
1180 vs 370C)
in
0D600-0.6 =
whencellsaremost
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, TITLE DATE
induction -
adding 5th to the culture
Autoinduction
↳ relies on consumption of a carbon source in the media
I cell's metabolic pathway uses another method for energy production
cell C source used up, removes repression promoter
↳ as grows, on
:allows protein expression
Example:Regulation of
it polymerase expression in E. coli expression before
inducer added
1
-
expression of recombinant proteins can be toxic excess production, leaky expression)
↳ prevent by separating gene cloning from gene expression
Phage i7 promoter
>
does not bind native E.coli
T7 polymerase
plasmidw/ target gene,
controlled by PTI promoter
bacteria are specially engineered
to carry the phage it polymerase gene >bind DT7 -> add inducer
->
drives target gene expression
DNA
polymerases used for PCR
high relative processivity efficient
=
* Taq is efficient but lacks 3'-5'
proofreading ability, adds A
overhang
at3'end
rest
* makes less product (lower yield)
but more correct products
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