Heterologous expression of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Triticum aestivum and Arabidopsis thaliana.
Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus cells and the pRSETB vector was successful for expression of a soluble His(6)-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.
Piattoni
Claudia V
CV
Instituto de Agrobiotecnología del Litoral (IAL, CONICET-UNL), Facultad de Bioquímica y Ciencias Biológicas, Paraje El Pozo, CC 242, S3000ZAA Santa Fe, Argentina.
Rius
Sebastián P
SP
Gomez-Casati
Diego F
DF
Guerrero
Sergio A
SA
Iglesias
Alberto A
AA
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D004586:Electrophoresis; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases