PHAGE CLUB: The biotechnological use and further development of phage display

• Forschungsstelle: EU FRP
• Projektnummer: 96.0046-1
• Projekttitel Englisch: PHAGE CLUB: The biotechnological use and further development of phage display

Schlüsselwörter (Englisch)

Phage; elisa; protein immobilization

Alternative Projektnummern (Englisch)

EU project number: ca. BIO4CT960389

Forschungsprogramme (Englisch)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Kurzbeschreibung (Englisch)

See abstract

Partner und Internationale Organisationen (Englisch)

SIK (SE) (co-ordinator)

Abstract (Englisch)

Rabbit monoclonal antibodies (RmAb) are not routinely obtained by eukaryotic cell fusion techniques. Therefore, we have applied the phage display technology to produce a recombinant rabbit Fab molecule directed against the KLH model antigen. The Fab fragments selected from the rabbit phage display library were subcloned in an expression vector to allow the production of a fusion protein comprising a dimer of bacterial alkaline phosphatase (phoA). This fusion protein is directly produced into the periplasmic space of E. coli. We show that a crude extract containing these conjugates can be used in a direct enzyme immunoassay as exemplified in the case of the KLH antigen.
Experiments are now under way to use large filamentous phage libraries expressing Ab molecules that had been made synthetically and with random variability. Initial experiments indicate that such libraries can be used to isolate specific mAb against a desired antigen provided the library has a large (>1010) Ab - variability.
To develop a system that allows efficient and stable indirect immobilization of a variety of recombinant multimeric proteins to solid phase, a new vector was constructed which allows the expression of up to three proteins linked by the Jun/Fos leucine zipper. Purification of the resulting protein was achieved by Ni+ affinity chromatography utilizing the 6xHis-ABP (albumin binding protein) protein fused to the N-terminus of the Jun polypeptide. The high binding affinity of ABP to rat serum albumin (RSA) was exploited for indirect immobilization of recombinant proteins to solid phase. In an enzyme linked assay, the binding of ABP to immobilized RSA was shown to be 100 to 1000 times more efficient than other immobilization systems. Using the ZZ IgG binding domain of staphylococcal protein A as bait, the RSA-ABP immobilization system was successfully used to screen and enrich lgG Fc encoding DNA fragments from a cDNA phage library. The newly designed vector termed pJuFoexpress allows production and purification of multimeric protein complexes linked by the Jun/Fos leucine zipper. Without chemical modifications, the recombinant proteins can be immobilized indirectly to solid phase. The immobilization results in the stable display of native, biologically active proteins that can be used in ELISA and phage display systems.

Datenbankreferenzen (Englisch)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 96.0046-1