| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
|---|---|---|
| Prev | Chapter 4. Materials and methods | Next |
To express Eci1p as a recombinant protein in E. coli, ECI1 was amplified from S. cerevisiae genomic DNA using PCR and gene-specific primers. Genomic DNA was isolated according to Ausubel and co-workers (1989) from BJ1991 cells. The PCR product was ligated into pUC plasmid (Pharmacia) and subsequently sequenced (Sanger et al. 1977) to verify the correct open reading frame. ECI1 was further subcloned into the pET3a expression vector (Novagen), and pET3a-ECI1 was transformed into the expression host, BL21(DE3)pLysS E. coli cells (Novagen).