c-terminus Archives - Gen9 Genetics
Summary: Phage display is a powerful technique that allows easy target identification. for any type of ligand. The targets are displayed on the surface of the phage as a fusion protein to a of the phage coat proteins. Through a repeated affinity selection process on a ligand, a specific enrichment of the displayed objectives will occur. In our studies using C-terminal display of cDNA fragments to the phage coat protein p6, we noted the occasional enrichment of targets that do not contain an open reading frame. This event has been previously described in other phage display studies using N-terminal display of targets for phage coat proteins and it was due to rare translation events such as frame shifting. The objective of this study was to examine whether the display of the C terminal of the lenses at p6 is also subject to frame shifting. For For this purpose, an enriched target was selected that did not contain an open reading frame and an E-tag.
attached to terminal C to measure target display on the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of measured lens and E-tag to detect occurrence of frame shift. As a As a result, we were able to demonstrate target visualization at both reading 0 and reading +1. frame indicating that the frame shift can also take place when the C-terminal fusion to lower coat protein p6 is applied.
Phage display is a high-throughput molecular technique that has been used successfully to select targets for any given ligand. Targets can be easily displayed on the surface of the phage virion by coupling the foreign DNA to a gene encoding a phage coat protein. After infection of the host, the phage The protein components are produced by the protein translation machinery of the infected bacterial cell and foreign DNA will show up as a fusion product to one of the phage coat proteins [1]. Because the physical link between genotype and phenotype, filamentous phage showing a relevant The polypeptide will be retained during affinity selections on candidate binding ligands followed by
target identification [2].
The most commonly used phage coat proteins for fusion are minor coat protein 3 (p3) and major protein coat protein 8 (p8) [3,4]. When using these coat proteins, the N-terminal fusion of the target is mandatory. for successful phage propagation. For visualization of cDNA libraries, N-terminal fusion is not possible due to the inherent stop codons present in the cDNA fragments, as depicted in Figure 1. However, the The free carboxyl terminus of the minor coat protein 6 (p6) allows successful fusion of the cDNA without interfering with phage propagation [5]. Using the phagemid vector pSP6 which was specifically designed to allow C-terminal fusion of targets to p6, we and others have already identified a variety of targets