reconstitution of the complexes from the purified, detergentsolubilized subunits is often not possible. Co-expression of the
subunits of a complex from one polycistronic messenger RNA
supports a balanced expression. Unexpectedly, E. coli operons
were shown to be translated with the proper stoichiometry even
when the subunit stoichiometry significantly deviates from one
copy each per complex [7]. Accordingly, the order of genes in a
synthetic operon needs to be experimentally optimized if the protein production is not balanced [8].
1.2.3 Recombinant
Membrane Protein
Expression
When expressing eukaryotic proteins in E. coli, codon optimization
of the corresponding gene is recommended in order to account for
the E. coli codon usage bias and thus avoid low or unreliable
expression. Overexpression of recombinant membrane proteins
can be optimized by fusing a green fluorescent protein (GFP) to
the membrane protein. Using fluorescence detection, GFP serves as
a marker for membrane protein localization, quantity of folded
protein, and the efficiency of solubilization from the membrane
by detergents [9, 10].
In this chapter, we describe the production of the E. coli holotranslocon, a seven-subunit membrane protein complex comprising
SecY, SecE, SecG, SecD, SecF, YajC, and YidC. It is one of the
largest recombinant membrane protein complexes produced to
date. The holo-translocon complex comprises all proteins known
to be important for protein translocation into and across the E. coli
plasma membrane [11, 12]. For the balanced expression of the
holo-translocon, the ACEMBL system was used [13, 14]. ACEMBL
comprises acceptor and donor vectors which have multi-integration
elements for individual genes or polycistrons. Donor vectors differ
from acceptor vectors and other commonly used plasmids in that
they comprise a conditional origin of replication (oriR6Kγ) and
therefore need to be propagated in strains with the phage R6Kγ
pir gene. All vectors have a LoxP site, but different antibiotic
resistances. Donor and acceptor vectors can be fused by Cre recombinase and the donor–acceptor fusion plasmid can be selected in a
conventional pir
¯ E. coli strain using the appropriate combination
of antibiotics [13].
For the expression of the holo-translocon, the conserved SecY,
SecE, and SecG subunits were encoded by a synthetic polycistronic
operon on a donor vector (Fig. 1a). A second donor vector carried
the gene for YajC, which is the only nonessential subunit of the
holo-translocon [15]. These two donor vectors were fused to an
acceptor vector with a polycistron encoding for YidC, SecD, and
SecF. Hexa-histidine tags were fused to YidC, SecD, and SecG and
a calmodulin-binding peptide to YajC (Fig. 1a). Double-affinity
purification via these tags results in pure holo-translocon complex,
comprising one copy of each subunit [15, 16].
6
Burak V. Kabasakal et al.
subunits of a complex from one polycistronic messenger RNA
supports a balanced expression. Unexpectedly, E. coli operons
were shown to be translated with the proper stoichiometry even
when the subunit stoichiometry significantly deviates from one
copy each per complex [7]. Accordingly, the order of genes in a
synthetic operon needs to be experimentally optimized if the protein production is not balanced [8].
1.2.3 Recombinant
Membrane Protein
Expression
When expressing eukaryotic proteins in E. coli, codon optimization
of the corresponding gene is recommended in order to account for
the E. coli codon usage bias and thus avoid low or unreliable
expression. Overexpression of recombinant membrane proteins
can be optimized by fusing a green fluorescent protein (GFP) to
the membrane protein. Using fluorescence detection, GFP serves as
a marker for membrane protein localization, quantity of folded
protein, and the efficiency of solubilization from the membrane
by detergents [9, 10].
In this chapter, we describe the production of the E. coli holotranslocon, a seven-subunit membrane protein complex comprising
SecY, SecE, SecG, SecD, SecF, YajC, and YidC. It is one of the
largest recombinant membrane protein complexes produced to
date. The holo-translocon complex comprises all proteins known
to be important for protein translocation into and across the E. coli
plasma membrane [11, 12]. For the balanced expression of the
holo-translocon, the ACEMBL system was used [13, 14]. ACEMBL
comprises acceptor and donor vectors which have multi-integration
elements for individual genes or polycistrons. Donor vectors differ
from acceptor vectors and other commonly used plasmids in that
they comprise a conditional origin of replication (oriR6Kγ) and
therefore need to be propagated in strains with the phage R6Kγ
pir gene. All vectors have a LoxP site, but different antibiotic
resistances. Donor and acceptor vectors can be fused by Cre recombinase and the donor–acceptor fusion plasmid can be selected in a
conventional pir
¯ E. coli strain using the appropriate combination
of antibiotics [13].
For the expression of the holo-translocon, the conserved SecY,
SecE, and SecG subunits were encoded by a synthetic polycistronic
operon on a donor vector (Fig. 1a). A second donor vector carried
the gene for YajC, which is the only nonessential subunit of the
holo-translocon [15]. These two donor vectors were fused to an
acceptor vector with a polycistron encoding for YidC, SecD, and
SecF. Hexa-histidine tags were fused to YidC, SecD, and SecG and
a calmodulin-binding peptide to YajC (Fig. 1a). Double-affinity
purification via these tags results in pure holo-translocon complex,
comprising one copy of each subunit [15, 16].
6
Burak V. Kabasakal et al.