NYMC Faculty Publications
A Novel Efficient Producer of Human ω-Amidase (Nit2) in Escherichia Coli
Author Type(s)
Faculty
DOI
10.1016/j.ab.2021.114332
Journal Title
Analytical Biochemistry
First Page
114332
Last Page
114332
Document Type
Article
Publication Date
11-1-2021
Department
Biochemistry and Molecular Biology
Abstract
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.
Recommended Citation
Epova, E., Shevelev, A. B., Shurubor, Y. I., Cooper, A. J., Biryukova, Y. K., Bogdanova, E. S., Tyno, Y., Lebedeva, A. A., & Krasnikov, B. F. (2021). A Novel Efficient Producer of Human ω-Amidase (Nit2) in Escherichia Coli. Analytical Biochemistry, 632, 114332-114332. https://doi.org/10.1016/j.ab.2021.114332
