potential+vectors+for+phage+display

NEB M13KE T7Select Phage Display

Letter to companies about phage display vector Novagen Stratagene Promega NEB

1-4-12 I could have checked Agilent as well. Andrey e-mailed me about this.

Kurt,

Can you check these sources: @http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=607

@http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=611

@http://www.progen.de/en/psex81-surface-expression-phagemid-vector.html

2010: Vector (pComb3XSS) (available from Carlos Barbas III, The Scripps Research Institute)

The first link is about the pBC phagemid vectors from Agilent @http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=607 f1 ori, pUC ori, chloramphenicol resistant It looks like these plasmids are unsuitable because the proteins are expressed as a fusion with lacZ instead of pIII

The second link is about the pBlueScript II Phagemid System from Agilent which the pBC phagemid vectors were derived from. @http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=611 Once again, the genes are expressed as a fusion with lacZ instead of pIII.

The third link is about pSEX81 from Progen Biotechnik which appears to be a German company. This vector actually looks very promising. @http://www.progen.de/en/psex81-surface-expression-phagemid-vector.html This is a phagemid vector. The vector is designed for producing scFvs as a fusion with pIII. It has an F1 and ColE1 ori. It is ampicillin resistant. Insertion Scheme: The whole scFv is inserted which has the following linker: "The VH and VL genes were joined by a DNA-fragment coding for a flexible 18 amino acid residue linker containing the first six amino acids of the CH1 constant region domain and the hydrophilic pig brain alpha-tubulin peptide sequence EEGEFSEAR." Note that this linker is referred to as the YoI tag. Cloning procedure: To clone VH gene fragments the recognition sites of the restriction endonucleases NcoI and HindIII are recommended. VL gene fragments should be introduced by using the recognition sites of the enzymes MluI and NotI. Message to Progen 1-5-12

The pComb3XSS vector is available from the Scripps Research Institute http://www.scripps.edu/mb/barbas/content/pcomb_images/updatepcomb_image.htm Maps http://www.scripps.edu/mb/barbas/content/pcomb_images/pcomb_images_files/pComb_Maps/pComb3X_Maps.pdf insert is a fusion with gene III, ampicillin resistance, f1 ori, pBR322 (not labeled in their "maps" file) Insertion scheme?: Message to Carlos at Scripps 1-5-12

What was the vector used by the group who made the helper plasmids I plan to use? It was a pDAN5 phagemid (see Helper Plasmid). pDan5 was actually created by Andrew Bradbury. This paper has a map of the plasmid Exploiting recombination in single bacteria to make large phage antibody libraries http://www.nature.com/nbt/journal/v18/n1/abs/nbt0100_75.html insert as fusion with pIII protein, M13 ori, colE1 ori, ampicillin resistance

What did my original PCANTAB5E phagemid look like? ampicillin resistant, insert expressed as fusion with pIII, pBR322 ori (in the same class as ColE1), f1 ori Insertion scheme: entire scFv inserted all together using SfiI and NotI at beginning of pIII gene. Linker between heavy and light chain?: (Gly4Ser)3

Qualities I would like in a vector for phage display
 * it should be a phagemid (I think; Kathy seems to prefer this)
 * should express insert as a fusion with pIII protein if I want to express it as an M13 phage
 * it would be great if it had SfiI and NotI sites, but this is not completely critical