Two-cistron system overexpression of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunit B and B-derivatives from spinach in Escherichia coli

A gene coding for the subunit B (GapB) of chloroplast glyceraldehyde-3-phos phate dehydrogenase fi om spinach and its two derivatives (GapBc) lacking t he GapB-specific C-terminal extension have been cloned by RT-PCR, These thr ee genes have been overexpressed with full activity in Escherichia coli whe n a two-cistron expression system controlled by an inducible promoter P,, i s used. With a suitable base composition of the first cistron, the expressi on level of GapB and the derivatives GapBc are expressed up to 15-20% of th e total cell protein and around 20 mg of recombinant GapBcs with full activ ity are purified from 1 liter of cultured bacteria. The specific activity o f the two derivatives GapBc (40-60 u/mg) is similar to that of GapA (50-70 u/mg) and lower than that of reported GapBc derivative (E, Baalmann, R, Sch eibe, R. Cerff, and W. Martin, 1996, Plant Mol. Biol. 32, 505-513). (C) 200 1 Academic Press.