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Development of an optimized refolding process for recombinant Ala-Glu-IGF-1
K R Hejnaes1, S Bayne, L Nørskov
1Hagedorn Research Laboratory, Gentofte, Denmark.
Protein Engineering
|December 1, 1992
Summary
Researchers optimized the in vitro folding of N-terminal extended insulin-like growth factor-1 (AE-IGF-1) by controlling redox potential, achieving a 60% yield. The folded protein was converted to IGF-1, with scrambled IGF-1 identified as a major byproduct.
Area of Science:
- Biochemistry
- Protein Chemistry
- Molecular Biology
Background:
- Insulin-like growth factor-1 (IGF-1) is a crucial growth-promoting hormone.
- Production of recombinant IGF-1 often involves refolding denatured protein.
Purpose of the Study:
- To optimize the in vitro folding of denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1).
- To investigate the factors influencing renaturation yield and disulfide bond formation.
- To characterize the major by-product formed during enzymatic conversion to IGF-1.
Main Methods:
- Purification of AE-IGF-1 from Escherichia coli extracts.
- In vitro folding under varying redox potentials.
- Enzymatic conversion of folded AE-IGF-1 to IGF-1.
- Identification of by-products using enzymatic digestion and molecular modeling.
Main Results:
- Renaturation yield was dependent on solution redox potential, with maximal yield of 60% (w/w) achieved.
- Scrambled IGF-1 was identified as a major by-product (20% w/w).
- Two potential disulfide bond arrangements were proposed for scrambled IGF-1, with one being energetically more stable.
Conclusions:
- Redox potential is a critical factor for efficient in vitro folding of AE-IGF-1.
- Scrambled IGF-1 represents a stable, yet undesired, conformation.
- Understanding disulfide bond formation is key to improving IGF-1 production.