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Protein expression is used to increase the abundance of a protein of interest within a cell or 'host,' to aid further study or experiments which may require massive quantities of the protein, such as structural studies. One of the most widely used hosts for expression experiments is E. coli, and the DNA that encodes the protein of interest is inserted into the E. coli in the for of a plasmid expression vector. Once inside the host, the expression vector can either increase the binding strength of the promoter region, thus helping the process of transcription or it can increase the copies of the gene present.
Even after a protein of interest has been over expressed within a host, isolation of this protein is still required. This is where protein purification techniques are used to separate the protein from a host or complex mixture. The separation of proteins is essential if the protein of significance is to be characterised in terms of its structure, interactions with other proteins and the function. A way in which this can be achieved is by the addition of a 'tag' onto the gene that encodes the protein and placing this into an expression vector. The vector allows the protein to fuse with the tag which then undergoes affinity chromatography which differs depending on the nature of the tag used.
Invitrogen's Gateway technology utilises the site-specific recombination properties of bacteriophages lamda. This results in a highly efficient and extremely rapid way to transport a gene of interest into a number of different vector systems that Gateway provides.
Cloning into an entry vector for the Gateway system
The initial stage required for entry into the Gateway system is the cloning of the gene of interest into an entry vector. In the journal entry vectors pDB and pDBHis with a TEV (tobacco etch virus) protease recognition site were used, however there are other entry vectors that can be used such as pENTR directional TOPO vectors (pENTR/D-TOPO and pENTR/SD-TOPO). Every entry vector for the Gateway system are designed so that the gene of interest is flanked by attL recombination sites which allows for the entry into the expression vectors at a later stage without the need for restriction enzymes and ligases. The exclusion of these restriction enzymes and ligases means that the entry into the vectors occurs within hours rather than days, whilst still performing to a high efficiency standard.
Within the Gateway system, the BL21-AIâ„¢ E. coli strain is used as the host for the expression. This contains "â€¦a chromosomal insertion of the gene encoding T7 RNA polymerase (T7 RNAP) into the araB locus of the araBAD operonâ€¦" Therefore the regulation of the T7 RNA polymerase gene is controlled by the araBAD promoter. The addition and concentration of the sugars, L-arabinose and glucose modulate the amount of expression or repression that occurs. The sugar, L-arabinose forms a complex with the transcriptional regulator AraC which causes other reactions that result in the release of the DNA loop and thus allows transcription to begin. If glucose is added to the solution, this represses the level of transcription by reducing the amount of the activator protein, cAMP.
The E. coli Expression System with GatewayÂ® Technology contains a series of
GatewayÂ®-adapted destination vectors designed to facilitate high-level, inducible
expression of recombinant proteins in E. coli using the pET system. Depending on
the vector chosen, the pDESTâ„¢ vectors allow production of native, N-terminal, or
C-terminal-tagged recombinant proteins (see table below).
For structural studies, high yields of soluble proteins
are required. Unfortunately, even when sequence information
is available, there is no clue to predict protein
behavior when produced in a given expression system.
Since the bacterium Escherichia coli is easy to handle, is
inexpensive, and grows quite fast, it is usually used as the
principal expression system [1,2]. Nevertheless, a large
fraction of proteins overexpressed in E. coli often accumulate
as inclusion bodies . Classical approaches for
increasing soluble recombinant protein expression in
E. coli cells are modiWcations of culture parameters such
as temperatures, additives, or even induction conditions.
However, not all proteins follow the same rules of heterologous
expression and many may require more drastic
modiWcations such as adding fusion partners or even
changing the expression system to be produced as soluble
proteins [4-7]. While classical approaches described
above do not require further subcloning to be carried
out, the comparison of fusion partners' impact on protein
solubility led to subcloning the ORF1 of interest in a
library of expression vectors that becomes laborious when handling a large number of genes. Recently,
Hartley and co-workers  have described a cloning
method (Gateway technology) that enables rapid cloning
of one or more genes into virtually any expression
vector using site-speciWc and conservative recombination,
eliminating the requirement to work with restriction
enzymes and ligase.
Here, we describe the construction and the applicability
of a new expression vector set for E. coli adapted to the
Gateway technology, encoding an N-terminal fusion and
a six-histidine tag either N or C terminal. In addition to
an ampicillin resistance marker and the ColE1 origin, the
vectors bear elements for T7-promoter-based expression
of recombinant proteins. Since transcription is driven by
T7 RNA polymerase tightly regulated by the lac operator
sequence, a parallel expression screening is amenable
using autoinductible medium (F. William Studier, Brookhaven
National Laboratory, personal communication).
The vectors, derived from the same backbone (pET22b;
Merck, Darmstadt, Germany), have been designed for (i)
a rapid parallel subcloning of any ORF sequence of interest
from any entry vector, (ii) a consistent comparison of
the impact of fusion partner(s) on protein expression and
solubility, and (iii) a choice of the six-histidine tag's
location for further aYnity puriWcation.
To validate the applicability, two genes encoding for
signaling proteins in Bacillus subtilis (SBGP codes E0508
and E0511) were cloned into the vector set and an
expression and solubility proWle was determined using
Coomassie-stained sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). Finally, translation
and accessibility of the six-histidine tag was
checked by purifying expressed soluble proteins using
immobilized metal aYnity chromatography (IMAC).