RESUMEN
Nit2/ω-amidase catalyzes the hydrolysis of α-ketoglutaramate (KGM, the α-keto acid analogue of glutamine) to α-ketoglutarate and ammonia. The enzyme also catalyzes the amide hydrolysis of monoamides of 4- and 5-C-dicarboxylates, including α-ketosuccinamate (KSM, the α-keto acid analogue of asparagine) and succinamate (SM). Here we describe an inexpensive procedure for high-yield expression of human Nit2 (hNit2) in Escherichia coli and purification of the expressed protein. This work includes: 1) the design of a genetic construct (pQE-Nit22) obtained from the previously described construct (pQE-Nit2) by replacing rare codons within an 81 bp-long DNA fragment "preferred" by E. coli near the translation initiation site; 2) methods for producing and maintaining the pQE-Nit22 construct; 3) purification of recombinant hNit2; and 4) activity measurements of the purified enzyme with KGM and SM. Important features of the hNit2 gene within the pQE-Nit22 construct are: 1) optimized codon composition, 2) the presence of an N-terminus His6 tag immediately after the initiating codon ATG (Met) that permits efficient purification of the end-product on a Ni-NTA-agarose column. We anticipate that the availability of high yield hNit2/ω-amidase will be helpful in elucidating the normal and pathological roles of this enzyme and in the design of specific inhibitors.
