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Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

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The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibrio cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.

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The possibility of improving the antibody responses to a model streptococcal antigen, administered by intranasal immunization as surface-displayed on the food-grade bacterium Staphylococcus carnosus, by co-exposure of a peptide (CTBp) comprising amino acids 50^75 of the cholera toxin B subunit, was investigated. It was found that the introduction of the CTBp into the chimeric surface proteins, containing a serum albumin binding protein (ABP) from streptococcal protein G as model antigen, significantly increased serum IgG responses upon intranasal immunization. Similarly, elicited local IgA responses were also found to be improved. Furthermore, it was demonstrated that live delivery of the staphylococci was required to obtain this effect, since UV-irradiated or heat-killed bacteria exposing the same chimeric surface proteins did not show increased anti-ABP IgG responses. ß