**2. Anchoring domains in surface display systems of LAB**

The cell surface anchoring domains utilized for the development of LAB surface display systems are associated with the cell surface either covalently or noncovalently. Depending on the applied anchoring domains, two modes of the protein display can be considered, including internal and external mode of the protein display. In the case of the internal mode of protein display, fusions of target proteins to anchoring domains are expressed in LAB cells, and therefore target proteins are displayed on the surface of expression hosts, whereas in the case of the external mode of protein display, expression and display hosts are different from each other. If the association of anchoring domains with the cell surface is of a non covalent nature, they can bind to cells when they are added from the outside. Therefore, fusions of target proteins to anchoring domains are produced in suitable expression hosts capable of their correct folding and modifications. The fusion proteins are then purified and incubated with desired display hosts in order to attach to the cell surface. As a result, the external mode of protein display can retain the nongenetically modified status of cells and is valuable for food and vaccine development.

The surface display systems based on the internal mode of protein display are often associated with the limitations in terms of the translocation of target proteins to the cell surface and the control of surface intensity of target proteins. The mislocalization of target proteins can affect their functions negatively( Dieye et al., 2003; Van Der Vaart et al., 1997; Wan et al., 2002). In contrast, the surface display systems based on the external mode of protein display can ensure the full exposure of target proteins outside of the cell wall and the surface intensity of target proteins can readily be adjusted by selecting appropriate display hosts and suitable concentrations for the fusion proteins in the incubation mixture. However, regarding noncovalent interaction of target proteins with the cell surface, the possibility of dissociation of target proteins from the cell surface should be considered.

## **2.1. Covalent anchors**

428 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

the gastrointestinal tract (GIT).

valuable for food and vaccine development.

LAB are gram positive, non-spore forming, fastidious, acid tolerant, and strictly fermentative that secret lactic acid as the major end product of sugar fermentations (Axelsson, 1998). LAB are naturally present in media rich in organic nutrients such as food products and digestive tracts. They are a genetically diverse group of bacteria with GC contents varying from 34 to 53%, including rod shaped bacteria such as lactobacilli and also cocci such as lactococci, enterococci, pediococci, and leuconostoc (Stiles & Holzapfel, 1997). Since time immemorial, LAB have been used for the fermentation and preservation of food products, particularly dairy products, fermented meats, and vegetables. Consequently, several strains of LAB have a long record of safe association with humans and human foodstuffs (Mckay & Baldwin, 1990). The display of proteins on the surface of LAB cells can broaden or improve applications of these bacteria. In this chapter, we intend to describe cell surface anchoring domains used in LAB surface display systems. Then applications of surface engineered LAB are depicted and key factors affecting their performances are highlighted. Moreover, we explained comprehensively a novel application of the protein display in LAB, which is potentially useful for enhancement of the delivery of viable LAB to

**2. Anchoring domains in surface display systems of LAB** 

The cell surface anchoring domains utilized for the development of LAB surface display systems are associated with the cell surface either covalently or noncovalently. Depending on the applied anchoring domains, two modes of the protein display can be considered, including internal and external mode of the protein display. In the case of the internal mode of protein display, fusions of target proteins to anchoring domains are expressed in LAB cells, and therefore target proteins are displayed on the surface of expression hosts, whereas in the case of the external mode of protein display, expression and display hosts are different from each other. If the association of anchoring domains with the cell surface is of a non covalent nature, they can bind to cells when they are added from the outside. Therefore, fusions of target proteins to anchoring domains are produced in suitable expression hosts capable of their correct folding and modifications. The fusion proteins are then purified and incubated with desired display hosts in order to attach to the cell surface. As a result, the external mode of protein display can retain the nongenetically modified status of cells and is

The surface display systems based on the internal mode of protein display are often associated with the limitations in terms of the translocation of target proteins to the cell surface and the control of surface intensity of target proteins. The mislocalization of target proteins can affect their functions negatively( Dieye et al., 2003; Van Der Vaart et al., 1997; Wan et al., 2002). In contrast, the surface display systems based on the external mode of protein display can ensure the full exposure of target proteins outside of the cell wall and the surface intensity of target proteins can readily be adjusted by selecting appropriate display hosts and suitable concentrations for the fusion proteins in the incubation mixture. However, regarding noncovalent interaction of target proteins with the cell surface, the possibility of dissociation of target proteins from the cell surface should be considered.
