T7Select® Phage Display Vectors
There are three basic types of T7Select® phage display vectors; the T7Select415-1 vector for high copy number display of peptides, the T7Select1-1 and -2 vectors for low copy number display of peptides or larger proteins, and the T7Select10-3 vector for mid copy number display of larger peptides and 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 amino acid 348 of the 10B capsid protein.
Functional peptides up to about 50 amino acids have been displayed from T7Select415. Expression of the T7Select415 capsid gene is controlled by the same strong phage promoter (l10) and translation initiation site (s10) as in wild-type phage. T7Select415 clones usually grow well on normal T7 laboratory hosts, such as E. coli BL21 (it does not grow on male strains). The capsid shell of the phage is composed entirely of the capsid/peptide fusion protein, thereby displaying 415 copies of the peptide on the surface of the phage. High copy number display is desirable wherever a strong signal is useful, such as in epitope mapping. It may also be important for obtaining peptides that bind with low affinity to their targets.
Functional proteins up to slightly more than 1000 amino acids have been displayed from T7Select1-1 vectors. The T7Select1-2a, b, c series provides multiple cloning sites in all three reading frames. Peptides or proteins are displayed in low copy number (about 0.1–1 per phage) from these vectors, which makes them suitable for the selection of proteins that bind strongly to their targets. To obtain low copy display, the promoter of the capsid gene and the translation initiation site were removed. The capsid mRNA is still produced from phage promoters located further upstream of the gene, but production of capsid protein is greatly reduced. T7Select1 phage are grown on a complementing host that provides large amounts of the 10A capsid protein from a plasmid clone. The 10A gene in the complementing plasmid and the capsid gene in the vectors have been engineered to minimize any recombination between the genes.
T7Select10-3 enables the display of an intermediate number of capsid fusions (5–15 per phage). To obtain mid copy display, a modified translation initiation site was added upstream of gene 10A in the low copy vector, which results in enhanced fusion protein production. The mid copy vector must be grown in host strains that supply complementing gene 10A product. The T7Select10-3 vector is ideal for constructing cDNA libraries because this vector represents a good overall balance between expression level and suitability for high affinity screening. Unlike other display systems that utilize N-terminal fusions, inserts are fused at the C-terminus of T7 gene 10, enabling the expression and display of inserts that contain internal stop codons. This is important when using oligo(dT) primed cDNA because poly(A) is usually downstream from a translation stop codon in mRNA.
The sequence is provided in GenBank format, with significant features indicated. To convert the sequence into other formats, first select the sequence and copy (Command-C on Mac or Ctrl-C on Windows). Next, click here to open the NIH ReadSeq web page in a new window. Paste the sequence into the input field (Command-V on Mac or Ctrl-V on PC), choose your favorite format from the dropdown menu, and hit the "submit" button.
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