Trends in Biotechnology
Microbial cell-surface display
Section snippets
Carrier protein (anchoring motif)
During the 1990s, many different carrier proteins (anchoring motifs) were developed for the surface display of proteins. The use of different carriers often results in different physiological effects on host cells. For example, using proteins that are essential for cellular functions or structures, such as outer membrane proteins and subunits of cellular appendages, might lead to growth defects and destabilization of cell envelope integrity. A successful carrier should meet the following four
Passenger protein (target protein)
The passenger protein to be displayed is selected by the required application. However, it should be noted that passenger proteins themselves also influence the translocation process and final surface display. Different passenger proteins fused to the same carrier protein are transported to the different locations in the same host strain [25]. The characteristics of passenger proteins are known to affect transportation process significantly. The folding structure of the passenger protein (such
Host strain
Selection of a host strain for surface display is an important factor that cannot be neglected. A good host should be compatible with the protein to be displayed and should be easy to cultivate without cell lysis. Also, the host strain should have low activities of cell wall associated and extracellular proteases. It was shown that cholera toxin B subunit (CtxB) was displayed successfully in an ompT (encoding the outer membrane protease T, OmpT) negative strain but was released into the medium
Surface display systems developed for Gram-negative bacteria
Gram-negative bacteria possess a complex cell envelope structure that consists of cytoplasmic membrane, periplasm and outer membrane. This means that the surface-anchoring motif, fused with the protein to be displayed, should pass through the cytoplasmic membrane and periplasm to the outer membrane. The targeting and anchoring mechanisms of carrier proteins vary among the different surface proteins and different approaches have been used to develop successful display systems. As shown in Table 1
Surface display systems developed for Gram-positive bacteria
Many surface proteins of Gram-positive bacteria are covalently immobilized to the cell wall, typically involving a specific C-terminal sorting signal consisting of 32–38 amino acids. Staphylococcal protein A (SpA) has often been used as a model system to study anchoring mechanisms of surface proteins in Gram-positive bacteria (Fig. 3a). The sorting signal includes an LPXTG (in single-letter amino acid code, where X denotes any amino acid) sequence motif followed by a stretch of ∼23 hydrophobic
Surface display systems developed for yeast
The display of foreign proteins on the surface of yeast provides several unique advantages. Two types of mannoproteins are present in the cell wall of Saccharomyces cerevisiae: sodium dodecyl sulfate (SDS)-extractable and glucanase-extractable mannoproteins. The SDS-extractable mannoproteins appear to be associated noncovalently with the cell wall and can be extracted from the cell wall by treating with SDS and a reducing agent, such as DTT or β-mercaptoethanol. The glucanase-extractable
A peek at the future development
Despite the successful development of various cell-surface display systems, problems remain to be solved and improvements to be made. For example, questions have been raised over the quality of the peptide library displayed on the cell surface because of the potential bias introduced by the sequence-dependent variation in expression level. In the development of whole-cell biocatalysts by cell-surface display, the reduction in the activity of enzyme is an issue. Compared with their free forms,
Acknowledgements
Our work described in this paper was supported by the National Research Laboratory program of the Ministry of Science and Technology, the Basic Industrial Research Program of the Korean Ministry of Commerce, Industry and Energy, and by the Center for Ultramicrochemical Process Systems (CUPS).
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