Proteins, cell surface display

In vivo methods for creating genotype-phenotype linkages are well established and exploit the cellular machinery necessary to process some proteins. Cell surface display of proteins coupled with fast cell-sorting systems can increase the size of the library that can be displayed and selected or screened. 

Figure 3.3 Cell surface display. Proteins displayed on the cell surface of organisms bind a fluorescent molecule. Cells are passed through a FACS machine, allowing separation into populations that do or do not bind the target...

Shimazu, M., Mulchandani, A. and Chen, W. (2001) Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnology Progress, 17, 76-80. 

Successful cell surface display of the protein can be verified by inducing gene expression via addition of anhydrotetracycline to the culture medium and immunofluorescence staining of the cells using an antibody directed against the protein to be displayed (Protocol 1). Analysis can be performed by fluorescence microscopy or flow cytometry. 

Ligand binding, and certain enzymatic assays, can be greatly simplified if a protein is biosynthetically anchored on the external surface of a cell. Surface displayed proteins are readily accessible to fluorescent probes and thus, transport limitations are circumvented. In addition,... 

Directed Evolution of Binding Proteins by Cell Surface Display Analysis of the Screening Process... 

Figure 8.5. In vivo methods allow connection between phenotype and genotype using cellular machinery. Synthesized proteins remain in the cytoplasm (or are secreted) in the nondisplay method. Cell surface display is a more recently introduced technique that uses special plasmids in order to produce proteins capable of transport into and retention by the cell membrane.

Fig. 2.5 The linking of phenotype and genotype in selection systems for antibodies. For more details on (a-c), see text, (a) In phage display, the antibody is fused to the minor coat protein g3p of filamentous phage, while the DNA is on the inside of the phage, (b) In yeast or bacterial cell surface display, the antibody is displayed on the outer surface of the cell, while the genetic information is encoded on a plasmid inside the cell, (c) In ribosome display, mRNA and protein product are linked by the ribosome, and the selection takes place in an in vitro translation system. Addition-...

Glycosylated-inositol (GPI)-anchored proteins have also been used to anchor foreign peptides for cell-surface display in S. cerevisiae [55] and Hansenula poly-morpha [56]. Four different gpi genes and their encoded proteins were characterized for the purpose of displaying fusion protein on the cell surface. The FLOl gene encodes the Flolp protein, a lectin-like cell-wall protein thought to form stemlike structures that cover the distance of the cell wall. This protein has also been used to express peptides on the yeast surface [57]. 

Kim S Y, Sohn J H, Pyun Y R, et al. (2002). A cell surface display system using novel GPI-anchored proteins in Hansenula polymorpha. Yeast. 19 1153-1163. 

Cell surface displayed recombinant or namral protein is used directly as the selection target... 

Synthetic biology aids metabolic engineering approaches and lifts the technology to a new level [25]. New biological parts generate new possibilities and many of them are tested in baker s yeast as common host. For example, cell-surface display of functional proteins opens an entirely new universe to engineer efficient cell factories. Tanaka and Kondo report about the combination of metabolic engineering... 

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