Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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Phagemid Display System pSKAN

Construction of Libraries with Enormous Molecular Diversity

Features

  • 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-pL-promoter

Applications

  • High-throughput screening of protein-protein interactions
  • Delection 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.

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.
Preparation of phagemid particles

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, ampicillin.) Note: Since M13 derived phagemids do not lyse the cells, colonies and not plaques are counted!

Vector map of pSKAN8

Vector map of pSKAN8

Phagemid vector construction

Phagemid vector construction.

pMAMPF vector produces the soluble variant of hPSTI protein.

Kit Components

Product Amount
HyA pSKAN Library in E. coli WK6ΛmutS 1 ml
HyB pSKAN Library in E. coli WK6ΛmutS 1 ml
HyC pSKAN Library in E. coli WK6ΛmutS 1 ml
Host strain E. coli WK6ΛmutS 1 ml

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
PSKAN pSKAN libraries (3) in E.coli, incl. E.coli strain WK6 lmutS 1 mL each 538,00 PDF
PS1255 sequencing primer # 1255 (18 mer), lyophilized 500 pm 109,00 PDF
PS2897 sequencing primer # 2897 (19 mer), lyophilized 500 pm 109,00 PDF
PSKAN3 anti P III (g3p) - antibody, monoclonal mouse 100 µl 241,00 PDF
PSKAN2 anti hPSTI - antibody (SPINK1), monoclonal mouse 100 µl 241,00 PDF
PSKAN8 pSKAN8 vector,lyophilized DNA 5 µg 233,00 PDF
PSKAN4 pMAMPF-3-PSTI4 vector, lyophilized DNA 5 µg 233,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

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Literature

Phagemid Display

  1. Maywald F, Boldicke T, Gross G, Frank R, Blocker H, Meyerhans A, Schwellnus K, Ebbers J, Bruns W, Reinhardt G, et al.(1988): Human pancreatic secretory trypsin inhibitor (PSTI) produced in active form and secreted from Escherichia coli. Gene. 1988 Sep 7;68(2):357-69. Pubmed
  2. Boldicke T, Kindt S, Maywald F, Fitzlaff G, Bocher M, Frank R, Collins J (1988): Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides. Eur J Biochem. 1988 Aug 1;175(2):259-64. Erratum in: Eur J Biochem 1989 Feb 15;179(3):725. Pubmed
  3. Collins J, Szardenings M, Maywald F, Blocker H, Frank R, Hecht HJ, Vasel B, Schomburg D, Fink E, Fritz H (1990): Human leukocyte elastase inhibitors: designed variants of human pancreatic secretory trypsin inhibitor (hPSTI). Biol Chem Hoppe Seyler. 1990 May;371 Suppl:29-36. Pubmed
  4. Szardenings M, Collins J (1990): A phasmid optimised for protein design projects: pMAMPF. Gene. 1990 Sep 28;94(1):1-7. Pubmed
  5. Paulsen J, Kroner KH, Schuette H, Szardenings M, Hustedt H (1989): Membrane and adsorption processes in the purification of the human pancreatic secretory trypsin inhibitor (hPSTI) Ber. Bunsenges. Phys. Chem. 93, 1015-1019(1989)
  6. Paulsen J. et al., DECHEMA Biotech. Conf., DECHEMA, Frankfurt, pp1031 (1989)
  7. Hecht HJ, Szardenings M, Collins J, Schomburg D (1991): Three-dimensional structure of the complexes between bovine chymotrypsinogen A and two recombinant variants of human pancreatic secretory trypsin inhibitor (Kazal-type). J Mol Biol. 1991 Aug 5;220(3):711-22. Pubmed
  8. Collins, J. et al., in Wooley et al. (ed.) D.F.I. Print, Aarhus, pp34 (1990)
  9. Klaus W, Schomburg D (1993): Solution structure of a variant of human pancreatic secretory trypsin inhibitor determined by nuclear magnetic resonance spectroscopy. J Mol Biol. 1993 Feb 5;229(3):695-706. Pubmed
  10. Hecht HJ, Szardenings M, Collins J, Schomburg D (1992): Three-dimensional structure of a recombinant variant of human pancreatic secretory trypsin inhibitor (Kazal type) J Mol Biol. 1992 Jun 20;225(4):1095-103. Pubmed
  11. Szardenings M, Vasel B, Hecht HJ, Collins J, Schomburg D. (1995): Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design. Protein Eng. 1995 Jan;8(1):45-52. Pubmed
  12. Roettgen P, Collins J (1995): A human pancreatic secretory trypsin inhibitor presenting a hypervariable highly constrained epitope via monovalent phagemid display. Gene. 1995 Oct 27;164(2):243-50. Pubmed
  13. Zell R, Fritz HJ (1987): DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J. 1987 Jun;6(6):1809-15. Pubmed Free
  14. Tesar M, Beckmann C, Rottgen P, Haase B, Faude U, Timmis KN (1995): Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunotechnology. 1995 May;1(1):53-64. Pubmed
  15. Beckmann C, Haase B, Timmis KN, Tesar M (1995): Multifunctional g3p-peptide tag for current phage display systems. J Immunol Methods. 1998 Mar 15;212(2):131-8. Pubmed
  16. Marks JD, Hoogenboom HR, Griffiths AD, Winter G (1992): Minireview: Molecular evolution of proteins on filamentous phage. Mimicking the strategy of the immune system. J Biol Chem. 1992 Aug 15;267(23):16007-10. Pubmed Free
  17. Dimasi N, Martin F, Volpari C, Brunetti M, Biasiol G, Altamura S, Cortese R, De Francesco R, Steinkuhler C, Sollazzo M (1997): Characterization of engineered hepatitis C virus NS3 protease inhibitors affinity selected from human pancreatic secretory trypsin inhibitor and minibody repertoires. J Virol. 1997 Oct;71(10):7461-9. Pubmed Free
  18. Mersmann M, Schmidt A, Tesar M, Schoneberg A, Welschof M, Kipriyanov S, Terness P, Little M, Pfizenmaier K, Moosmayer D (1998): Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay. J Immunol Methods. 1998 Nov 1;220(1-2):51-8. Pubmed
  19. 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
  20. 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
  21. 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

References with the pSKAN System

  1. 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
  2. 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
  3. Noda K, Yamasaki R, Hironaka Y, Kitagawa A (2001): Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library. FEMS Microbiol Lett. 2001 Dec 18;205(2):349-54. Pubmed
  4. 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
  5. 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
  6. 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