High-throughput screening of surface displayed gene products

Area of Science:

• Molecular Biology
• Biotechnology
• Immunology

Background:

• The completion of the human genome project necessitates functional analysis of gene products.
• Automated technologies are crucial for characterizing proteins, their expression, and localization, maintaining a link to encoding genes.
• Phage display offers a complementary approach for recombinant protein expression and selection of binding molecules.

Purpose of the Study:

• To develop and implement automated high-throughput technology for phage display library handling and selection.
• To establish a system for simultaneous screening of libraries against diverse target molecules.
• To select human single-chain variable fragment (scFv) antibody fragments against human cDNA expression products.

Main Methods:

• Development of automated high-throughput technology using picking-spotting robots and magnetic particle handling.
• Utilizing filamentous bacteriophage or phage mid vectors for cloning, creating a physical link between protein and DNA.
• Immobilization of target molecules on high-density filter membranes or magnetic particles for screening.
• Confirmation of binding activity using magnetic particle ELISA in a microtitre format.

Main Results:

• Successful implementation of an automated system for high-throughput phage display library handling and selection.
• Demonstrated ability to screen libraries simultaneously against different target molecules.
• Selection of human scFv antibody fragments against expression products of human cDNA libraries.

Conclusions:

• The developed automated phage display system enables efficient and high-throughput selection of binding molecules.
• This technology facilitates the functional analysis of gene products by linking expressed proteins to their encoding DNA.
• The system is fully automatable from target molecule immobilization to confirmed binder clones, advancing recombinant protein and antibody fragment selection.