T7Select® phage display system, Novagen®
Leverantör: Merck Millipore (Novagen)
Novagen® T7Select® Phage Display System takes advantage of the properties of bacteriophage T7. This system is easy to use and has the capacity to display peptides up to about 50 amino acids in size in high copy number (415 per phage), and peptides or proteins up to about 1200 amino acids in low copy number (0,1 to 1 per phage) or mid-copy number (5 to 15 per phage).
Phage assembly takes place in the E. coli cytoplasm and mature phage are released by cell lysis. Unlike the filamentous systems, peptides or proteins displayed on the surface of T7 do not need to be capable of secretion through the cell membrane, a necessary step in filamentous phage assembly.
T7 has additional properties that make it an attractive display vector. It is very easy to grow and replicates more rapidly than either bacteriophage l or filamentous phage. Plaques form within 3 hours at 37 °C and cultures lyse 1 to 2 hours after infection, decreasing the time needed to perform the multiple rounds of growth usually required for selection. The T7 phage particle is extremely robust, and is stable to harsh conditions that inactivate other phage. This stability expands the variety of agents that can be used in biopanning selection procedures, which require that the phage remain infective. T7 is an excellent general cloning vector.
The T7Select® Phage Display System uses the T7 capsid protein to display peptides or proteins on the surface of the phage. The capsid protein is normally made in two forms, 10A (344 aa) and 10B (397 aa). 10B is produced by a translational frameshift at amino acid 341 of 10A, and normally makes up about 10% of the capsid protein. However, functional capsids can be composed entirely of either 10A or 10B, or of various ratios of the proteins. This finding provided the initial suggestion that the T7 capsid shell could accommodate variation, and that the region of the capsid protein unique to 10B might be on the surface of the phage and could be used for phage display.
There are 3 basic types of T7Select phage display vectors: the T7Select415 vector for high-copy number display of peptides, the T7Select10 vector for mid-copy number display of peptides or larger proteins, and the T7Select1 vectors for low-copy number display of peptides or larger proteins. In all of the vectors, coding sequences for the peptides or proteins to be displayed are cloned within a series of multiple cloning sites following aa 348 of the 10B protein. The natural translational frameshift site within the capsid gene has been removed, so only a single form of capsid protein is made from these vectors.
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