Biomedical Engineering Reference
In-Depth Information
1.2.3 Secretion into
the Periplasmic Space
in E. coli
For the production of recombinant proteins that are normally secreted
(from either bacterial or eukaryotic sources), there are a number
of advantages in targeting them for periplasmic secretion in
E. coli
.
Disulphide bond formation is favored in the periplasm, protease
activity is lower than in the cytoplasm, and there are fewer host
proteins, all of which facilitate recovery of the product. Gram-negative
bacteria have a number of different secretory pathways that direct
export of proteins to different destinations, such as the periplasmic
space and outer membrane [
37
,
38
]. The most commonly used
mechanisms for secretion of heterologous proteins in
E. coli
are the
Type I and Type II pathways [
39
,
40
]. In Type I secretion, the pro-
tein is transported in one step across the inner and outer membranes
and is released into the media [
41
]. By contrast, in Type II secretion
the protein is exported via a two-step mechanism. There are three
different mechanisms, namely the SecB, signal recognition particle
(SRP), and TAT pathways [
40
]. The SecB and TAT systems translo-
cate proteins post-translationally with the latter system requiring the
presence of specifi c factors in the cytoplasm [
40
]. By contrast the SRP
pathway involves the co-translational translocation of the protein.
There is evidence that heterologous proteins may be handled differ-
ently by the SRP and SecB pathways. Higher levels of production of
recombinant DARPins (small scaffold binding proteins) were observed
when the proteins were targeted via the SRP mechanism compared to
the SecB mechanism [
42
].
We have designed a suite of fi ve vectors that target two of
the Type II secretory mechanisms. The vectors provide both an
N-terminal signal sequence (three are designed to target the SecB
mechanism and two the SRP mechanism) and a C-terminal hexa-
histidine tag (Table
4
). Figure
4
shows the results from our stan-
dard Ni
2+
-NTA expression screen for expression of the New Delhi
Metallo-
-lactamase 1 (NDM-1) from
Klebsiella pneumoniae
;
the cytoplasmically expressed version of the construct with a
C-terminal is shown for comparison. Interestingly in Rosetta2
(DE3) plysS we see strong expression from all fi ve signal sequences
and only very low levels of the cytoplasmically expressed version of
the protein. Moreover in B834 (DE3) we see the same low levels
of cytoplasmic expression but only see strong expression from the
PelB leader suggesting the choice of screening strain may be an
important variable in the periplasmic secretion of heterologous
proteins.
β
2
Materials
1. The homology regions required for In-Fusion™ cloning are
generated by adding extensions to both forward and reverse
PCR primers. The sequence and length of the extensions
are determined by the type of restriction endonuclease used to
2.1 Primers
and Vectors
