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Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity
Published on: May 1, 2018
Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats
Kritika Pershad1, Mark A Sullivan, Brian K Kay
1Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Avenue, Molecular Biology Research Building, Room 4318, Laboratory for Molecular Biology (M/C 567), Chicago, IL 60607, USA.
Analytical Biochemistry
|February 15, 2011
Summary
Researchers developed a novel "drop-out" phagemid vector to streamline the process of subcloning affinity reagents. This new vector simplifies the transfer of phage display inserts, saving time and effort in downstream screening and characterization.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Engineering
Background:
- Phage display is a common method for generating affinity reagents.
- Subcloning these reagents into expression vectors is a laborious and time-consuming step.
- Efficient downstream characterization is crucial for validating affinity reagents.
Purpose of the Study:
- To develop a simplified and rapid method for converting phage display vectors into expression vectors.
- To reduce the time and effort associated with subcloning affinity reagents.
- To create a versatile vector system for generating various affinity reagent fusion proteins.
Main Methods:
- Construction of a novel "drop-out" phagemid vector.
- Utilized restriction enzyme digestion (MfeI and AscI) to sequentially remove gene III and alkaline phosphatase (AP) coding sequences.
- Generated C-terminal six-histidine tagged affinity reagents.
- Validated the vector system using four human single chain Fragments of variable regions (scFv).
Main Results:
- The "drop-out" phagemid vector efficiently converts to expression vectors via simple restriction digestion.
- Gene III and AP drop-out efficiency ranged from 90% to 100% for tested constructs.
- Successfully generated alkaline phosphatase and six-histidine tagged scFv fusions.
- Demonstrated specific binding of scFv reagents to target proteins including zebrafish (Danio rerio) proteins (suppression of tumorigenicity 13, recoverin, Ppib) and human Lactoferrin.
Conclusions:
- The developed "drop-out" phagemid vector significantly speeds up the downstream screening of affinity reagents.
- This vector system bypasses the need for laborious and time-consuming subcloning experiments.
- It offers a versatile and efficient platform for generating diverse affinity reagent fusion proteins for biochemical characterization and screening.
