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Published on: January 17, 2015
Efficient bacterial production of functional antibody fragments using a phagemid vector
Hiroyoshi Kuba1, Atsuko Furukawa, Toshihide Okajima
1Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Protein Expression and Purification
|December 14, 2007
Summary
This study introduces a novel bacterial expression system for engineering antibody fragments (Fab). Optimized conditions enable high-yield production of correctly folded, antigen-binding Fab proteins without helper phage.
Area of Science:
- Biotechnology
- Protein Engineering
- Molecular Biology
Background:
- Phage display is used for antibody fragment (Fab) protein engineering via random mutagenesis and biopanning.
- Engineered Fab proteins may exhibit poor folding and bacterial production efficiency due to high-affinity mutations.
Purpose of the Study:
- To develop an efficient bacterial expression system for producing engineered Fab proteins.
- To overcome low folding efficiency in engineered antibody fragments.
- To achieve high-yield extracellular secretion of functional Fab proteins.
Main Methods:
- A direct bacterial expression system for phagemid-coded Fab proteins was established, eliminating the need for helper phage.
- Optimized bacterial cultivation conditions, including extended cultivation time (>50 h) and low temperature (25°C), were determined.
- Random mutagenesis and antigen-based biopanning were employed for protein engineering.
Main Results:
- The new system achieved high yields of extracellularly secreted Fab proteins, ranging from 3-15 mg/L of culture.
- Optimized expression conditions significantly improved the folding efficiency of engineered Fab proteins.
- Purified Fab proteins demonstrated correct folding and antigen-binding capabilities.
Conclusions:
- The developed bacterial expression system enables efficient production of functional, engineered antibody fragments.
- Optimized cultivation strategies are crucial for overcoming folding challenges in protein engineering.
- This method offers a valuable tool for producing high-affinity antibody fragments for therapeutic and diagnostic applications.
