George A. Ricca

Modulation of expression of the human gamma interferon gene in E.coli by site-directed mutagenesis

Plasmids expressing 2 forms of human immune interferon (IFN-gamma) in E. coli have been constructed: 1) pIFNTacI which expresses IFN-gamma with an N-terminal amino acid sequence of met-cys-tyr-cys-gln-, and 2) pIFNTacII which is a derivative of pIFNTacI from which the 9 base pairs (bp) coding for the cys-tyr-cys have been deleted. Quantitation of Western blots showed that approximately 10-fold more IFN-gamma was produced in cells harboring pIFNTacII (7.5% of total cellular protein) as compared to pIFNTacI. The IFN-gamma expressed in E. coli pIFNTacII is biologically active and routinely recoverable at 10(9) units per liter. When examined microscopically, IPTG induced E. coli harboring either plasmid construction contains prominent cytoplasmic inclusion bodies.

Cloning, expression of the human substance K receptor, and analysis of its role in mitogenesis.

The primary strudure of the human substance K receptor was established from the sequences of complementary DNA clones isolated from a human jejunal complementary DNA library. It consists of 398 amino acids, including seven putative transmembrane regions. The gene for the human substance K receptor was localized to chromosome region 10p13-10q23, a region with frequent chromosomal abnormalities. The human substance K receptor was expressed in transfeded NIH-3T3 cells lacking endogenous substance K receptors, and Scatchard analysis of 125I labeled substance K binding indicates approximately 100,000 receptors/cell with a single dissociation constant of 12 nM. Covalent cross-linking experiments utilizing 125I-substanceK and three different chemical cross-linking reagents (disuccinimidyl suberate, disuccinimidyl tartrate, or 1-ethyl-3-(3dimethylaminopropyl)carbodiimide-HCI) demonstrate an apparent molecular weight of 45,000, consistent with little or no N-linked glycosylation. The binding of substance K to its receptor on transfeded cells led to a rapid increase in the produdion of total inositol phosphates and the release of Ca2� from internal stores. Growth of the cells transfeded with the human substance K receptor is stimulated by the addition of substance K to the medium to a level similar to 10% serum. Therefore, the human substance K receptor can function as a growth fador receptor when expressed in mouse 3T3 cells.