pC3C Phage Display Plasmids

Phagemid pC3C for phage display of Fab with human constant domains and accompanying plasmid pC3C-His for the E. coli expression of Fab with a C-terminal hexa-histidine tag.

Highlights:

  • Yield 100 ug to 2 mg of protein for downstream evaluation and characterization of Fab binding properties
  • Only 2 PCR steps required for library assembly (compared to 3 PCR steps for pComb3H and pComb3X phage display vectors)
  • pC3C-His version contains hexa-tag for easy Fab purification

The Fab molecule was the first generated antibody fragment and still dominates basic research and clinical applications. New phage display vectors were designed to generate and select Fab libraries with human constant domains. These vectors facilitate bacterial expression of human humanized and chimeric nonhuman/human Fab antibody fragments. They differ from currently available pComb3H and pComb3X phage display vectors by assembling human and chimeric nonhuman/human Fab libraries in two rather than three PCR steps. As a result these novel constructs retain the initial variable light and heavy chain sequences and improve the resulting Fab library's complexity in terms of number diversity and affinity. These constructs were developed with and without a His tag and yield approximately 100 ug to 2 mg of protein which can be used for evaluation and characterization of Fab binding properties such as affinity and specificity. Notably the His tag provides a handle to easily purify Fab.

From the laboratory of Christoph Rader, PhD, National Cancer Institute/NIH.

Catalog Number Product Size AVAILABILITY Price Qty
ENH068
pC3C-His Plasmid
250ng In stock
Price: $66.00
ENH067
pC3C Phage Display Plasmid
250ng In stock
Price: $66.00
Specifications
Product Type: Plasmid
Gene/insert name: VL-CL-VH cassette of a Fab that binds human GPA33
Organism: chimeric rabbit/human
Fusion Tag(s): hemagglutinin decapeptide (YPYDVPDYAS), pC3C-His also contains C-termnial hexa-his tag
Antibiotic Resistance: Ampicillin, carbenicillin
Buffer: Water
Grow in E. coli at 37 C: Yes
Cloning Site 5': SfiI GGCCcaggcGGCC
Cloning Site 3': SfiI GGCCccgtcGGCC
Insert Size: ca. 1,150 bp
Vector Backbone and Size: 4,706 bp
High or low copy: Low
Storage: -20C
Shipped: Room Temperature
Provider
From the laboratory of Christoph Rader, PhD, National Cancer Institute/NIH.
References
  1. Hofer T, Tangkeangsirisin W, Kennedy MG, Mage RG, Raiker SJ, Venkatesh K, Lee H, Giger RJ, Rader C. Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity with an improved phage display vector. J Immunol Methods. 2007 Jan 10;318(1-2):75-87.
  2. Kwong KY, Baskar S, Zhang H, Mackall CL, Rader C. Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity. J Mol Biol. 2008 Dec 31;384(5):1143-56.
  3. Rader C. Generation and selection of rabbit antibody libraries by phage display. Methods Mol Biol. 2009;525:101-28, xiv.
  4. Kwong KY, Rader C. E. coli expression and purification of Fab antibody fragments. Curr Protoc Protein Sci. 2009 Feb;Chapter 6:Unit 6.10.
  5. Baskar S, Suschak JM, Samija I, Srinivasan R, Childs RW, Pavletic SZ, Bishop MR, Rader C. A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display. Blood. 2009 Nov 12;114(20):4494-502.
  6. Stahl SJ, Watts NR, Rader C, DiMattia MA, Mage RG, Palmer I, Kaufman JD, Grimes JM, Stuart DI, Steven AC, Wingfield PT. Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev. J Mol Biol. 2010 Apr 2;397(3):697-708.
  7. Yang J, Baskar S, Kwong KY, Kennedy MG, Wiestner A, Rader C. Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PLoS One. 2011;6(6):e21018.
  8. Rader C. Generation of human Fab libraries for phage display. Methods Mol Biol. 2012;901:53-79.
  9. Rader C. Selection of human Fab libraries by phage display. Methods Mol Biol. 2012;901:81-99.
  10. Grönwall C, Charles ED, Dustin LB, Rader C, Silverman GJ. Selection of apoptotic cell specific human antibodies from adult bone marrow. PLoS One. 2014 Apr 23;9(4):e95999.
  11. Peng H, Nerreter T, Chang J, Qi J, Li X, Karunadharma P, Martinez GJ, Fallahi M, Soden J, Freeth J, Beerli RR, Grawunder U, Hudecek M, Rader C. Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility. J Mol Biol. 2017 Sep 15;429(19):2954-2973. doi: 10.1016/j.jmb.2017.08.003.
  12. Zhuang X, Watts NR, Palmer IW, Kaufman JD, Dearborn AD, Trenbeath JL, Eren E, Steven AC, Rader C, Wingfield PT. Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy. J Biol Chem. 2017 Oct 6;292(40):16760-16772. doi: 10.1074/jbc.M117.802272. Epub 2017 Aug 23.

If you publish research with this product, please let us know so we can cite your paper.

 
Loading...
Loading...