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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.

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.

Secretion of functional recombinant murine interleukin-2 (mIL2) by Lactococcus lactis was achieved by fusion of the sequence encoding mature mIL2 to the secretion signal leader of the lactococcal usp45 gene placed under transcriptional control of the phage T7 promoter-T7 RNA polymerase expression system. The recombinant mature mIL2 was one of only a few proteins which accumulated in the growth medium. Sequence analysis revealed correct processing at the first amino acid of the mature protein. A T-cell proliferation assay showed that the recombinant protein has the same specific biological activity as mIL2 obtained from a natural source.

Crystals of bacteriophage MS2 have been obtained by slowly cooling a 1% virus solution from 23 degrees C to 0 degrees C in the presence of poly(ethylene glycol) 6000. The crystals were colorless, needle-like, anisotropic and very fragile. Electron microscopic observation of the crystals revealed a two-dimensional lattice of particles with RNA phage morphology and dimensions. Preliminary X-ray examination of the crystals confirmed their viral nature.