1. High-Level Expression, Purification, and Renaturation of Recombinant Murine Interleukin-2 from Escherichia coli
- Author
-
Jan Demolder, Erik Remaut, Johan Robbens, Nico Mertens, Alex Raeymaekers, Walter Fiers, Joël Vandekerckhove, Geert Plaetinck, and Yves Guisez
- Subjects
Protein Denaturation ,Protein Folding ,Molecular Sequence Data ,Size-exclusion chromatography ,Gene Expression ,Biology ,medicine.disease_cause ,Inclusion bodies ,law.invention ,Mice ,law ,Complementary DNA ,Gene expression ,Escherichia coli ,medicine ,Animals ,Amino Acid Sequence ,Inclusion Bodies ,Messenger RNA ,Base Sequence ,Molecular biology ,Recombinant Proteins ,Chaotropic agent ,Solubility ,Biochemistry ,Recombinant DNA ,Interleukin-2 ,Plasmids ,Biotechnology - Abstract
A murine interleukin-2 (mIL-2)-encoding cDNA, isolated from a stimulated EL4 mRNA library, was used to construct several expression plasmids directing synthesis of the mature protein in Escherichia coli. The expression was under control of either the PTrp or the PL promoter. Using these systems, a high-level expression of between 10 and 35% of the total cellular protein was obtained. The mIL-2 protein, present as insoluble inclusion bodies, could be solubilized in a chaotropic mixture and was partially purified by preparative gel filtration under denaturing conditions. After renaturation, the protein was further purified to homogeneity by anion-exchange chromatography. Depending on the fermentation, induction, and renaturation conditions, the yield ranged between 0.35 and 1 mg of purified mIL-2/g wet cells. The specific biological activity was about 10(7) units/mg and the endotoxin content < 4 ng/mg pure recombinant protein.
- Published
- 1993