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Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization
Published on: December 23, 2015
Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth
1Howard P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Biotechnology Progress
|December 2, 2000
Summary
This study introduces a mini-intein for efficient protein purification, streamlining downstream processing. This controllable self-splicing element optimizes protein recovery in batch and flow systems.
Area of Science:
- Biotechnology
- Protein Engineering
- Biochemical Engineering
Background:
- Current fusion-based affinity separations face limitations in downstream processing.
- Efficient protein recovery is crucial for biotechnological applications.
Purpose of the Study:
- To optimize protein recovery using a controllable self-splicing mini-intein.
- To enhance both batch and flow purification strategies.
Main Methods:
- A tripartite fusion system incorporating a mini-intein linker was designed.
- Recombinant human acidic fibroblast growth factor (aFGF) served as the model protein.
- Mass transport modeling and kinetic studies (pH, temperature) were employed.
Main Results:
- The mini-intein facilitated controllable protein recovery, reducing purification steps.
- A mass transport model accurately predicted product concentration and peak shape.
- Temperature and pH influenced cleavage kinetics, providing insights into regulation.
Conclusions:
- Mini-inteins offer a viable strategy for streamlined and efficient protein purification.
- The developed system demonstrates broad applicability in protein recovery processes.
- Understanding cleavage kinetics enables further optimization of purification strategies.
