pSKAN libraries (3) in E.coli, incl. E.coli strain WK6 lmutS

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-pL-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.