ReviewExpressing genes in different Escherichia coli compartments
Introduction
The Gram-negative bacterium Escherichia coli remains the most versatile host for the production of heterologous proteins [1]. Although for most applications it is desirable to achieve maximal production within the cytoplasm, targeting the protein to extracellular compartments may offer an interesting alternative, especially when cytoplasmic production results in toxicity or improper folding [1]. Cell-surface display, using outer membrane proteins as carriers for epitopes, adhesins or metal-binding motifs has been the object of intense research during the past decade and has been reviewed recently 2, 3. In view of the attractive applications of surface display in different areas, including vaccine development, bioremediation and enzyme immobilisation, it is not surprising that intensive research in this domain has been ongoing during the past two years, leading to new and interesting developments. The intention of this review is to re-actualise the state of the art concerning the production of proteins in different Gram-negative bacterial compartments. Although Gram-positive bacteria can also be engineered to display proteins 3, this review will be limited to Gram-negative bacteria, in particular E. coli.
Section snippets
Soluble proteins: cytoplasm or periplasm?
The formation of disulfide bonds does not occur in the reducing environment of the cytoplasm because of the presence of thioredoxins. The paradigm of this reality is the enzyme alkaline phosphatase, which is active only in the periplasm 4. It therefore became an accepted fact that proteins with disulfide bonds could only be properly folded in the periplasm. It is now evident, however, that multiple disulfide bonds can be formed in the E. coli cytoplasm of a trxB thioredoxin reductase mutant,
Porins
Fig. 1 shows the different strategies that have been used to display both short peptides and large proteins on the surface of E. coli. The features of each system are summarised in Table 1.
Insertion of short amino-acid stretches can be achieved in extracellular loops of outer membrane proteins such as the maltoporin LamB 8, 9, 10, 11, 12, 13, OmpS of Vibrio cholerae 14 or OmpC of E. coli [15]. The applications of this technique range from the display of synthetic metal-binding motifs [8, 12, 15•
Secretion of heterologous proteins
The secretion of recombinant proteins, resulting in their liberation in the culture medium, is often desirable for easy recovery and purification. Of the four different secretion systems that have been described in Gram-negative bacteria (reviewed in 37), only type I, the Sec-independent ATP-binding cassette (ABC)-transporter-mediated transport system, has been readily engineered to secrete passenger proteins. The prototype of type I secretion systems is the hemolysin transport system, which
Conclusions
During the past few years, much progress has been made to improve the production of proteins in E. coli, not only in the cytoplasm, but also in other cellular compartments. It seems now that periplasmic expression should be limited to products that are toxic when present in the cytoplasm, as proteins with disulfide bridges can now be correctly folded in the cytoplasm by manipulating the thioredoxin pathway [5••, 6].
Targeting proteins to the outer membrane is now achievable thanks to a range of
Update
Since submission of this review, two important contributions in the domain of surface display were published. One concerns the display of the metal-binding metallothionein at the surface of the Gram-negative bacterium Ralstonia eutropha using the Neisseria gonorrhoeae IgA protease autotransporter [50], while the second one describes the display of hepatitis B and C at the surface of Salmonella typhi using the P. syringae Inp 51.
Acknowledgements
We wish to thank Mark Lauwerijs and A Leitão for reading and comments, Yves Geunes and Marc Vauterin for solving some computer problems, and the journal staff for their help.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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