Cloning, Sequencing and Expression of the atpE Gene of Broad Bean Chloroplast

The ninth KpnI fragment (about 4.0 kb) of broad bean chloroplast DNA contains the entire chloroplast atpE gene and atpB gene. This fragment was inserted into the pUC18 polylinker region, yielding plasmid pUK1. Southern hybridization using atpE gene from maize chloroplast as probe revealed that atpE gene encoding epsilon subunit of broad bean chloroplast was localized in 0.9 kb ClaI fragment of pUK1. The whole sequence of atpE gene of broad bean chloroplast was determined by using primer F and primer R of the polylinker region of pBluescript KS (+) DNA. The entire atpE gene of broad bean chloroplast was inserted into the polylinker region of vector pJLA505 to form recombinant plasmids pJLA505-atpE. The expression plasmid was transformed into E. coli DH5alpha cells which were then induced at 42 degrees. By analysis with SDS-PAGE, the product of interest accounted for more than 38% of total E. coli cell proteins. Identification of the expressed product by Western blot analysis demonstrated that it can react specifically with anti-epsilon serum of maize chloroplast CF(1). The expressed product aggregated to form insoluble inclusion bodies and was purified. The purified product had the same function as that of the native epsilon subunit.