Production of soluble and active recombinant murine interleukin-2 in Escherichia coli: high level expression, Kil-induced release, and purification.
We describe the production of soluble murine interleukin-2 (mIL2) and its purification following regulated release in the growth medium of Escherichia coli. The system is based on the ability of the Kil protein of pMB9 to release periplasmic proteins into the growth medium. As the kil gene is under control of the strong, but well regulatable pL promoter, the kil bearing plasmid is stably maintained in the cell. mIL2, fused to the outer membrane protein A (OmpA) signal peptide, was secreted into the periplasm and subsequently released into the growth medium after induction of the kil gene. This strategy allows a quick and easy purification of the heterologous protein without using strong denaturants or detergents, yielding a native protein with a specific biological activity equal to the natural mIL2. The system permits the production of mIL2 at levels up to 16 mg/liter. From a 12-liter fermentation, a final yield of about 30 mg of pure mIL2 was obtained.
Laboratory of Molecular Biology, Universiteit Gent, Belgium.
J
J
Robbens
A
A
Raeymaekers
L
L
Steidler
W
W
Fiers
E
E
Remaut
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007376:Interleukin-2; D011994:Recombinant Proteins; C069003:kil protein, E coli