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Abstract
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.
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