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Phagemid Display

Exclusively at MoBiTec!

Phagemid Display System pSKAN

Features and Advantages:

easy-to-handle
fast (results in less than 3 weeks!)
low cost
lab animals or tissue cultures become superfluous
direct physical link between genotype and phenotype: DNA codes for addressable tag on phage surface
construction of libraries with enormous molecular diversity
pSKAN phagemid targets small, recessed epitopes: "a fingertip instead of a whole hand" (antibodies)
stability of libraries due to strong repression of lambda-p_L-promoter

Applications:

high-throughput screening of protein-protein interactions
selection of proteins with specific binding properties (high affinity binders)
protease inhibitor screening
development of affinity matrices
ligand screening
development of pharmaceuticals and diagnostics
detection of allergens
of special interest: 3-D structure-based protein design

pSKAN phage presenting a hypervariable peptide loop on its surface. Enlarged: phage coat protein pIII is fused to the protein hPSTI (human pancreatic secretory trypsin inhibitor), which contains the hypervariable loop.

Introduction:

The pSKAN Phagemid Display System is a fast and easy to handle alternative to antibodies or immunoglobulin domain presenting phagemids. Since the pSKAN phagemid displays a small hypervariable amino acid loop, more than 3 x 10⁷ variants can be tested for the specific binding to a target molecule of interest. The variants with the optimal affinity can be selected and propagated without having to use animals or tissue cultures. The sequence of the binding domain can be determined easily by DNA sequencing. In comparison to antibodies, the pSKAN System is able to target very small epitopes, since it searches for binding sites with "only a fingertip" instead of "two whole hands" like antibodies. Within a peroid of only three weeks, the scientist is able to receive the target molecule of interest.

Panning with microplates. One well per library (HyA, HyB, HyC) for each reaction condition (# 1 - 4) is coated with the ligand of interest. After 3 - 5 rounds an increase in the pSKAN phagemid titer is observed. The control without panning target shows the background of unspecifically binding phagemids. (Amp, ampi-cillin.) Note: Since M13 derived phagemids do not lyse the cells, colonies and not plaques are counted! Below: pSKAN phagemid life cycle in E. coli (squares).

Product Description:

The pSKAN Phagemid Display System allows selection and production of novel proteins with the desired affinities to a given epitope. pSKAN is a combinatorial phagemid library expressed in E. coli: The phage coat protein pIII is fused to human pancreatic secretory trypsin inhibitor protein (hPSTI), which contains a short stretch of hypervariable amino acids. This loop is exposed and displays about 3 x 10⁷ randomly recombined amino acid variants. They can be screened in a "panning assay" for specific binding to a target molecule of interest. Variants with optimal affinity are selected and propagated in E. coli.
To determine the sequence of a selected hypervariable loop, two sequencing primers are available.
MoBiTec provides three pSKAN libraries which differ in the extent of their hypervariable loop: HyA, HyB and HyC.
Antibodies against pIII and hPSTI can be obtained for western blots. For the production of soluble hPSTI protein with the desired binding properties, the vector pMAMPF3-PSTI4 can be used. To construct your own peptide library, pSKAN8 vector DNA is available.

Phagemid vector construction.

pMAMPF vector produces the soluble variant of hPSTI protein.

For a detailed data sheet as a pdf-file, please click on the product No.

ORDER INFORMATION, SHIPPING STORAGE:
order#	description	amount
PSKAN	pSKAN Phagemid Display System (details see above): HyA, HyB, HyC pSKAN libraries incl. host strain shipped on dry ice; store at -70°C	3 x 1 ml 1 ml
PS1255	18 ^mer -sequencing primer 1255 shipped at RT; store at 4°C	500 pmole
PS2897	19 ^mer -sequencing primer 2897 shipped at RT; store at 4°C	500 pmole
PSKAN3	pIII antibody (monoclonal mouse IgG₁) shipped on blue ice; store at -20°C	100 µl
PSKAN2	anti-hPSTI antibody (monoclonal mouse IgG₁) shipped on blue ice; store at -20°C	100 µl
PSKAN8	pSKAN8 vector DNA, lyophilized shipped at RT; store at 4°C	5 µg
PSKAN4	pMAMPF3-PSTI4 vector DNA, lyophilized shipped at RT; store at 4°C	5 µg

See chapter 2 of our online catalog.

Download:

Handbook pSKAN & pSKAN8,
pSKAN 8 sequence

Literature

Ballard VL, Holm JM, Edelberg JM (2006): A quantitative PCR-based approach for rapid phage display analysis: a foundation for high throughput vascular proteomic profiling
Physiol Genomics. 2006 May 16; [Epub ahead of print] Pubmed
Cai D, Holm JM, Duignan IJ, Zheng J, Xaymardan M, Chin A, Ballard VL, Bella JN, Edelberg JM (2006): BDNF-mediated enhancement of inflammation and injury in the aging heart.
Physiol Genomics. 2006 Feb 14;24(3):191-7. Epub 2005 Dec 13. Pubmed Free
Kirsch M, Zaman M, Meier D, Dubel S, Hust M (2005): Parameters affecting the display of antibodies on phage.
J Immunol Methods. 2005 Jun;301(1-2):173-85. Pubmed
Edelberg JM, Wong A, Holm JM, Xaymardan M, Duignan I, Chin A, Kizer JR, Cai D (2004): Phage display identification of age-associated TNFalpha-mediated cardiac oxidative induction.
Physiol Genomics. 2004 Aug 11;18(3):255-60. Pubmed Free
Cai D, Xaymardan M, Holm JM, Zheng J, Kizer JR, Edelberg JM (2003): Age-associated impairment in TNF-alpha cardioprotection from myocardial infarction.
Am J Physiol Heart Circ Physiol. 2003 Aug;285(2):H463-9. Epub 2003 May 1. Pubmed Free
Verhaert RM, Van Duin J, Quax WJ (1999): Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd.
Biochem J. 1999 Sep 1;342 ( Pt 2):415-22. Pubmed
Hengerer A, Kosslinger C, Decker J, Hauck S, Queitsch I, Wolf H, Dubel S (1999): Determination of phage antibody affinities to antigen by a microbalance sensor system.
Biotechniques. 1999 May;26(5):956-60, 962, 964. Pubmed
Hengerer A, Decker J, Prohaska E, Hauck S, Kosslinger C, Wolf H (1999): Quartz crystal microbalance (QCM) as a device for the screening of phage libraries.
Biosens Bioelectron. 1999 Feb;14(2):139-44. Pubmed