Overexpression of the potential herbicide target sedoheptulose-1,7-bisphosphatase from Spinacia oleracea in transgenic tobacco

Several proteins are recalcitrant to expression in Escherichia coli. To explore transgenic plants as an alternative expression system, the gene encoding the potential herbicide target sedoheptulose-1, 7-bisphosphatase (SBPase, EC 3.1.3.37) was expressed in transgenic tobacco (Nicotiana tabaccum) under the control of a duplicated CaMV 35S RNA promoter. The active protein, a key enzyme in the Calvin cycle, accumulated to approximately 1.2% of total soluble protein. In order to purify recombinant SBPase, a sequence encoding six histidine residues was inserted C-terminally which allows a one step purification via Ni2+-NTA affinity chromatography. N-terminal amino acid sequence analysis of the purified protein confirmed processing of the transit peptide and revealed the previously unknown cleavage site. The transit peptide consists of 67 amino acids followed by the mature SBPase subunit of 342 amino acids including the C-terminal fusion. Purified SBPase was found to be enzymatically active after reduction with DTT and showed many biochemical properties of the native enzyme such as the dependence on Mg2+ and a pH optimum of 8.3. Subsequently, SBPase produced in transgenic tobacco was used in large-scale screening for the discovery of novel herbicides.