FIG. 5.
Production of full-length cALS by protein trans-splicing. Whole-cell lysates were prepared from uninduced E. coli ER2744 cells (lane 1) or from cells transformed with plasmids expressing cALS (lane 2), cALS(N)-INn (lane 3), INc-cALS(C) (lane 4), or cALS(N)-INn and INc-cALS(C) (lane 5) and were induced with 0.3 mM IPTG for 12 h at 25°C. Immunoblots were prepared after SDS-PAGE and were probed with antibodies against the N-terminal peptide (A) or C-terminal peptide (B) of cALS. A nonspecific 60-kDa protein species was detected by the antibody against the N terminus (A). The product of trans-splicing (cALS-14) has a slightly higher molecular weight than cALS because 14 additional amino acids are inserted at the splicing junction. kD, kilodaltons.