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-phosphate dehydrogenase from spinach and its two derivatives (GapBc) lacking the GapB-specific C-terminal extension have been cloned by RT-PCR. These three genes have been overexpressed with full activity in Escherichia coli when a two-cistron expression system controlled by an inducible promoter P(trc) is used. With a suitable base composition of the first cistron, the expression level of GapB and the derivatives GapBc are expressed up to 15-20% of the total cell protein and around 20 mg of recombinant GapBcs with full activity are purified from 1 liter of cultured bacteria. The specific activity of 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. Scheibe, R. Cerff, and W. Martin, 1996, Plant Mol. Biol. 32, 505-513).
Tang
G L
GL
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Fenglin Road, 354, Shanghai 200032, People's Republic of China.
Wang
Y F
YF
Bao
J S
JS
Chen
H B
HB
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005796:Genes / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018724:Spinacia oleracea / Q000166:cytology / Q000201:enzymology* / Q000235:genetics
D021122:Protein Subunits; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases