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Recombinant human glucagon: large-scale purification and biochemical characterization
K Yoshikawa1, H Tsuzuki, M Fujimoto
1Shionogi Research Laboratories, Shionogi and Co., Ltd., Osaka, Japan.
Journal of Protein Chemistry
|October 1, 1992
Summary
This study presents a scalable method for producing pure recombinant glucagon using Escherichia coli. The resulting glucagon is biochemically identical to pancreatic glucagon, demonstrating its therapeutic potential.
Area of Science:
- Biotechnology
- Protein Expression
- Biochemistry
Background:
- Recombinant glucagon production is crucial for therapeutic applications.
- Previous methods for recombinant glucagon expression have been reported.
- A scalable and efficient purification process is needed.
Purpose of the Study:
- To develop a large-scale method for isolating and purifying recombinant glucagon.
- To confirm the biochemical identity and purity of the produced recombinant glucagon.
Main Methods:
- Recombinant glucagon expressed as a fusion protein in Escherichia coli.
- Cell disruption, solubilization with guanidine-HCl, and digestion with Staphylococcus aureus V8 protease.
- Purification using S-Sepharose and Sephacryl S-100 chromatography, followed by analytical HPLC.
Main Results:
- A scalable purification method yielded approximately 180 mg of pure glucagon per liter of culture.
- Analytical HPLC confirmed glucagon purity of not less than 99.5%.
- Biochemical assays (radioreceptor assay, cAMP production, glycogenolysis) showed identity with pancreatic glucagon.
Conclusions:
- The developed method enables large-scale production of highly pure recombinant glucagon.
- Recombinant glucagon produced is biochemically identical to native pancreatic glucagon.
- This recombinant glucagon holds promise for therapeutic use.