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Optimization of Protein
Expression in E.coli
Optimization of protein expression in E.
coli can be exerted at the various levels outlined below:
Transcriptional control
can involve the following:
-
genetic fusion to strong promoters:
The level of transcription of a gene depends mostly on the frequency with
which the RNA polymerase initiates transcription. This, in turn, is a function
of the promoter sequence that is necessary and sufficient to allow
transcription by RNA polymerase. A standard E.
coli promoter is about a 40 bp region having 2 consensus sequences
centered around positions -10 and -35 from the transcription start site.
Examples of strong promoters include T7
RNA polymerase, lac, tac, pL).
Translational control
can involve the following:
- optimal spacing between ribosome-binding site and
initiation condon: A strong vector will have a ribosomal binding site
coding sequence (RBS) otherwise known as the
Shine-Dalgarno sequence, spaced an optimal distance (7-9 bp) from the initiation
codon (ATG). The Shine-Dalgarno sequence also should not be sequestered in
secondary structures nor should it overlap with a sequence recognized by an
RNA-binding protein.
- use of preferred codons: Codons vary from
organisms to organism so expressing a eukaryotic gene may use different codons
from that which your expression host uses. Bacterial hosts can
be used which encode tRNA genes corresponding to rare codons in E. coli. Such
codons are designed to enhance the expression of eukaryotic proteins that
contain codons rarely used in E. coli.
Post translational control
can involve the following:
Hosts like E. coli do not have the machinery for
posttranslational modifications as with eukaryotes. Thus, when expressing
certain eukaryotic genes it may be necessary to use eukaryotic systems like
yeast. However, there have been recent attempts to use E.coli to express postranscriptionally modified proteins.
A new strategy for the synthesis of glycoproteins.
Science.
2004 Jan 16;303(5656):371-3.
Other controls
for effective protein expression include:
There are a number of
problems that one can run into when cloning and expressing your protein of
interest. Here are a few of them as well as strategies you can use to try and
overcome these problems:
-
Toxic genes are
genes of interest which you introduce into your expression system (e.g., E.
Coli) which are toxic to your system. A toxic gene must be cloned and
maintained in the repressed state until its expression is desired. This could
be done for example using a T7 based expression vector which can be induced
with IPTG.
-
Premature termination
occurs when the RNA polymerase does not transcribe the entire message. One way
to deal with this is to use vectors which utilize strong anti-termination
systems to overcome transcription stop signals.
-
mRNA instability can
be dealt with by several strategies. 1) modify the sequence between the promoter
and the coding region to change the overall secondary structure of the
message, 2) use hosts with mutations in the genes encoding RNas since mRNA
turnover appears to involve exonucleases, 3) include strong terminators at the
end of the cloned gene in order to increase the message half-life.
-
Purification of your
recombinant protein: There are a number of ways that you can deal with
this. One way is to use tags on the amino terminal side of expressed proteins
such as:
(1) 6 histidine residues (6xHis). The
stretch of six histidine residues can be captured with a nickel ion that is
tightly bound to a nitrilotriacetic acid (NTA) matrix and then eluted.
(2) glutathione S-transferase (GST) tags
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