potential vectors for phage display
2014-08-29azim58 - 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.
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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
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2010: Vector (pComb3XSS) (available from Carlos Barbas III, The Scripps
Research Institute)
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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/pC
omb_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
- it would be great if it had SfiI and NotI sites, but this is not