slowed down by the requirement to optimize protein expression
and to subsequently identify conditions to solubilize the membrane
proteins without compromising integrity and activity. While the
optimal expression and purification conditions needs to be optimized for each new protein and complex, standard protocols as
presented here offer a good starting point for subsequent optimization by variation of conditions.
1.2 Recombinant
Expression
in Escherichia coli
E. coli has traditionally been, and still is, the most popular expression system for proteins. This is because of low costs, low expenditure of time, and the fact that it is less labor-intense than other
expression systems.
1.2.1 Choice of Promoter
and Strain
When expressing membrane proteins, saturation of the E. coli
membrane protein translocation and folding machinery should be
avoided. This would lead to protein misfolding, aggregation, and
formation of inclusion bodies. Very strong promoters such as the
T7 promoter frequently lead to these problems. Weaker promoters
synthesize membrane proteins at more moderate levels. Examples
include T7/lac hybrid promoters or the tightly regulated arabinose
promoter, both of which are often used for membrane protein
expression [2].
Addressing the same issues, tailor-made strains have been
developed for membrane protein production in E. coli. The T7
RNA polymerase-based expression strain BL21λ(DE3) and its
mutant strains C41λ(DE3) and C43λ(DE3) [3] are the most popular strains for expressing membrane proteins in E. coli [2]. C41
and C43 contain mutations in the lacUV5 promoter region of the
gene encoding for T7 RNA polymerase leading to lower expression
of the T7 RNA polymerase [4] and consequently slower transcription and translation rates of the proteins under the control of T7
promoter. The same principle applies to the E. coli BL21-AI strain
(Invitrogen) which expresses T7 RNA polymerase under the control of a tightly regulated arabinose promoter.
1.2.2 Other
Considerations
Auto-induction media are commonly used for improved membrane
protein production [5]. They can be used for all IPTG-inducible
expression systems. Protein production is induced in autoinduction media at high cell density when the glucose in the
media is depleted, which is during the mid/late log phase of
growth. Lactose uptake then leads to the production of allolactose
which causes the release of the lac repressor and induction of T7
RNA polymerase from the lacUV5 promoter.
For membrane protein complexes, a common strategy is the
co-expression of the proteins from their natural or artificial operons
[6] (see example in Fig. 1a). Co-expression of all subunits of a
complex allows complex assembly in vivo in the cell. This is particularly important for membrane protein complexes, where
4
Burak V. Kabasakal et al.
and to subsequently identify conditions to solubilize the membrane
proteins without compromising integrity and activity. While the
optimal expression and purification conditions needs to be optimized for each new protein and complex, standard protocols as
presented here offer a good starting point for subsequent optimization by variation of conditions.
1.2 Recombinant
Expression
in Escherichia coli
E. coli has traditionally been, and still is, the most popular expression system for proteins. This is because of low costs, low expenditure of time, and the fact that it is less labor-intense than other
expression systems.
1.2.1 Choice of Promoter
and Strain
When expressing membrane proteins, saturation of the E. coli
membrane protein translocation and folding machinery should be
avoided. This would lead to protein misfolding, aggregation, and
formation of inclusion bodies. Very strong promoters such as the
T7 promoter frequently lead to these problems. Weaker promoters
synthesize membrane proteins at more moderate levels. Examples
include T7/lac hybrid promoters or the tightly regulated arabinose
promoter, both of which are often used for membrane protein
expression [2].
Addressing the same issues, tailor-made strains have been
developed for membrane protein production in E. coli. The T7
RNA polymerase-based expression strain BL21λ(DE3) and its
mutant strains C41λ(DE3) and C43λ(DE3) [3] are the most popular strains for expressing membrane proteins in E. coli [2]. C41
and C43 contain mutations in the lacUV5 promoter region of the
gene encoding for T7 RNA polymerase leading to lower expression
of the T7 RNA polymerase [4] and consequently slower transcription and translation rates of the proteins under the control of T7
promoter. The same principle applies to the E. coli BL21-AI strain
(Invitrogen) which expresses T7 RNA polymerase under the control of a tightly regulated arabinose promoter.
1.2.2 Other
Considerations
Auto-induction media are commonly used for improved membrane
protein production [5]. They can be used for all IPTG-inducible
expression systems. Protein production is induced in autoinduction media at high cell density when the glucose in the
media is depleted, which is during the mid/late log phase of
growth. Lactose uptake then leads to the production of allolactose
which causes the release of the lac repressor and induction of T7
RNA polymerase from the lacUV5 promoter.
For membrane protein complexes, a common strategy is the
co-expression of the proteins from their natural or artificial operons
[6] (see example in Fig. 1a). Co-expression of all subunits of a
complex allows complex assembly in vivo in the cell. This is particularly important for membrane protein complexes, where
4
Burak V. Kabasakal et al.