Phage display of functional, full-length human and viral membrane proteins
Sudipta Majumdar, Agnes Hajduczki, Aaron S Mendez, Gregory A Weiss, Sudipta Majumdar, Agnes Hajduczki, Aaron S Mendez, Gregory A Weiss
Abstract
Phage display of protein and peptide libraries offers a powerful technology for the selection and isolation of ligands and receptors. To date, the technique has been considered limited to soluble, non-membrane proteins. We report two examples of phage display of full-length, folded and functional membrane proteins. Consistent display required the recently reported KO7(+) helper phage. The two proteins, full-length caveolin-1 and HIV gp41, display well on the surface of the phage, and maintain their binding activities as shown by in vitro assays.
Figures
![Figure 1](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/2597205/bin/nihms-68903-f0001.jpg)
Schematic representation of caveolin membrane topology and function.
![Figure 2](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/2597205/bin/nihms-68903-f0002.jpg)
Diagram of the phage ELISA experiment. Anti-FLAG antibody coated on the plate binds to a FLAG epitope on the N-terminus of the phage-displayed protein. An anti-P8, HRP-conjugated antibody binds specifically to the phage.
![Figure 3](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/2597205/bin/nihms-68903-f0003.jpg)
Phage display of two full-length membrane proteins. Using the format shown in Figure 2, display of caveolin or gp41 is assayed by binding to a FLAG epitope fused to the N-terminus of the protein. (A) Full-length human caveolin displayed on phage. Data points for caveolin phage binding to the negative control, BSA, overlay with data for the negative control KO7+ (no protein displayed on KO7+). All error bars indicate standard error (n=3 with the exception of 3B where n=2). (B) Full-length trimeric gp41 displayed on phage, assayed by an analogous method.
![Figure 4](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/2597205/bin/nihms-68903-f0004.jpg)
Phage-displayed, full-length caveolin binds to the gp41 ectodomain. In a format similar to the diagram in Figure 2, binding between phage-displayed caveolin and gp41 is assessed by ELISA targeting plate-bound gp41. High display levels of caveolin were demonstrated by anti-FLAG ELISA (data shown in graphical abstract).
Source: PubMed