Overexpression in E.coli of the regulatory subunit of protein kinase a from Saccharomyces cerevisiae

MORENO, S; PAVETO C; ROSSI, S; VARONE;C; ZAREMBERG, V

FASEB JOURNAL

FEDERATION AMER SOC EXP BIOL

Lugar: Bethesda; Año: 1997 vol. 11

0892-6638

AbstractOur aim was to train the students of a basic Molecular Biology coursr into the principles of DNA recombinanttechnology. This laboratory experiment is designed to last the who!´1 semester. In order lo obtain a fusion protein inframe with GST, the full length regulatory (R) subimit frnrn S .crrt ri.sioc-was subcloned in PGEX-4T :J. The codingregion of R was amplified by POU with suitable primers with EcoR 1 and Xho f flanking sequences from a Mockpiasmid containing the R sequence. The students désignée! the primers individualy as an exercise. P(´R fragment (1.2kb) and vector were cut with KcoK I and Xho I and ligated in different molar relationships. K.coli XLl-Blue wastransformed with the li gat ion products ami selected with ampiciiline. Adequate controls were included. Result-, atihis step were extensively discussed with the students. The screening was achieved by colony hybridi/ation using a nonradioactive probe, obtained by nick translation of the PCH fragment. Positive clones were analyzed by piasmidrestriction mapping and tested the production of the fusion protein after IF 1C! induction. The 70 kl)a fusion proteinwas visualized in SDS-PAGE and its functionality was revealed in crude extracts by the [3H]cAMP binding assay. I´hiswork was supported by torching grants from the UBA.