A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

Jarmander Johan, Gustavsson Martin, Do Thi-Huyen, Samuelson Patrik, Larsson, Larsson Gen

Description

The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli . We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His 6 -tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His 6 -tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His 6 -tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

Sujets

• Aïda
• Escherichia coli
• Proteolysis

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Jarmander et al. Microbial Cell Factories 2012, 11:118
http://www.microbialcellfactories.com/content/11/1/118
RESEARCH Open Access
A dual tag system for facilitated detection of
surface expressed proteins in Escherichia coli
1 1 2 1 1*Johan Jarmander , Martin Gustavsson , Thi-Huyen Do , Patrik Samuelson and Gen Larsson
Abstract
Background: The discovery of the autotransporter family has provided a mechanism for surface expression of
proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport
system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was
successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined
due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to
construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are
sensitive to proteolysis or otherwise difficult to express.
Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger
protein. Successful of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled
antibodies specific for the N-terminal His -tag and the C-terminal Myc-tag. While both tags were detected during6
SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage
of this protein that removes the His -tag facing the medium.6
Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly
used vector. The presence of the Myc- but not of the His -tag on the cell surface during H:gm expression allowed6
us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell
exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The
size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the
C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in
the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed
that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.
Keywords: AIDA, Surface expression, Autotransport, Escherichia coli, Proteolysis, Detection tag
Background shouldtheoretically besimplertouse thanGram-negative,
Surface expression of recombinant proteins was first as the expressed protein needs to be translocated over
described more than 25 years ago [1,2]. Systems for sur- only one cell membrane instead of the two required with
face expression in both Gram-negative and Gram- Gram-negative bacteria. However, since there is extensive
positive bacteria have been reported with a broad range documented knowledge regarding the genetics, growth
of applications in molecular biology, biochemistry, bio- and protein production of E. coli,itisanattractiveplat-
technology, microbiology and vaccinology [3-7]. The form also for surface expression applications. Laboratory
Gram-negative bacterium Escherichia coli has historic- strains of E. coli have the additional advantage that they
ally been one of the most extensively used hosts for lack inherent surface protein transporters, so there is less
recombinant protein production [8]. For surface expres- background of natural proteins on the cell surface and in
sion of heterologous proteins, Gram-positive bacteria the medium.
The discovery of the type V protein secretion pathway
* Correspondence: [e-mail] and of the autotransporter family [9] has provided great
1
School of Biotechnology, Division of Bioprocess Technology, Royal Institute of opportunities for surface expression of proteins in E.
Technology, KTH, Stockholm, SE 106 91, Sweden
coli. The surface translocation system of AIDA-I, theFull list of author information is available at the end of the article
© 2012 Jarmander et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.Jarmander et al. Microbial Cell Factories 2012, 11:118 Page 2 of 10
http://www.microbialcellfactories.com/content/11/1/118
C
adhesin involved in diffuse adherence of enteropatho- AIDA translocation unit, for a total size of 63 kDa not
genic E. coli, has for example been successfully trans- including the passenger (Figure 1 left, DNA sequence in
ferred to laboratory strains of E. coli [10] and used for Additional file 1). After the signal peptide had been
expression of recombinant proteins [11], enzymes [12] cleaved off, the fusion protein had a mature size of
and enzyme inhibitors [13] on the cell exterior. Auto- 58 kDa in the outer membrane. The previously used sur-
transporters have a conserved sequence organization face expression vector pDT1 [15] had functional units of
consisting of an N-terminal signal peptide followed by the same size and translated a protein of 62 kDa with a
the passenger protein, a linker region and a C-terminal mature size of 57 kDa in the membrane. The main fea-
translocation unit [14]. In the translocation process, the ture of pAIDA1 is that the translated fusion protein con-
signal peptide is cleaved off after a Sec-mediated passage tains two detection tags flanking the passenger on each
over the inner membrane. The widely accepted hypoth- side, compared to the protein product of pDT1, which
esis is that the C-terminal translocation unit then inserts has only one tag on the passenger’s N-terminal side
itself into the outer membrane and forms a β-barrel pore (Figure 1 right).
through which the linker and passenger protein pass and In pAIDA1, a His -tag was placed on the N-terminal6
become surface exposed. side of the passenger, as was previously done in pDT1.
We recently reported the display of the Salmonella The redundant nucleotides found in the gap between the
enterica serovar Enteritidis (S. Enteritidis) flagellar pro- tag and the passenger were however removed, and a
tein H:gm and fimbrial protein SefA on the surface of E. cleavage site for the recombinant type 14 3C protease
coli, using the AIDA-I autotransport system [15]. The from human rhinovirus (HRV 3C, aa seq: LEALFQGP)
proteins were expressed as fusions with an N-terminal was instead introduced in this space. On the C-terminal
His -tag to remove the need for protein specific anti- side of the passenger, a Myc-tag [16] was introduced. In6
bodies for detection of the passenger. In the vector that this case, a protease cleavage was also placed between
was used, the cleavage site for releasing the natural pas- the tag and the passenger, for theTobacco etchvirus pro-
senger from the cell surface had been removed, and the tease (TEV, aa seq: ENLYFQG). Since only relative sur-
fate of the expressed proteins were to stay covalently face expression levels can be measured by the detection
bound to the translocation unit. The aim of that study of tags, the introduction of protease cleavage sites makes
was to explore the possibility of using E. coli as a plat- it possible to develop a method for quantifying surface
form for presenting surface-exposed antigens. SefA was expression through conventional protein analysis.
successfully displayed on the cell surface, but the orienta- The natural promoter of AIDA-I, aidA, that was used
tion of H:gm in the outer cell membrane could not be in pDT1, transcribes mRNA constitutively and is regu-
resolved due to cleavage and loss of the His -tag. The hy- lated by a mechanism that is not well understood [17].6
pothesis that both fusion proteins were facing the cell ex- In pAIDA1, it was therefore replaced by the lacUV5 pro-
terior could not therefore be experimentally confirmed. moter, which is a well-studied, inducible promoter that
The present work aimed to create a vector with a dual can be successfully titrated in lacY negative E. coli
tag surface expression system, where two tags flank the strains to desirable expression levels [18]. In order to
passenger, in order to increase the possibility of detect- facilitate the modification of the novel vector, unique re-
ing proteins that are sensitive to proteolysis or difficult striction sites were placed between each component in
to translocate. We wished to use this vector to confirm the expression cassette so that each unit could be
the previous hypothesis that the H:gm fusion protein did manipulated independently.
indeed have the correct orientation in the outer mem-
brane, establishing the vector as an improved surface ex-
pression analysis tool. As the new vector leads in Surface expression of proteins
principle to the expression of a new fusion protein, we E. coli cells harboring pAIDA1 and pDT1 expressing the
also wished to compare the relative expression levels fusion proteins of SefA and H:gm were cultivated in
with those obtained with the previously used surface ex- batch, and cell samples from the logarithmic growth
pression system. SefA was chosen as the model protein phase were removed for analysis. The mature fusion pro-
for this comparison as it had earlier been successfully teins had the