Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase.
The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real M. tuberculosis NAD(+) synthetase. These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.
Dipartimento di Genetica e Microbiologia, Universitá di Pavia, Via Ferrata 1, 27100 Pavia, Italy. marco.b@ipvgen.unipv.it
Marco
M
Bellinzoni
Edda
E
De Rossi
Manuela
M
Branzoni
Anna
A
Milano
Fiorenzo A
FA
Peverali
Menico
M
Rizzi
Giovanna
G
Riccardi
D019732:Amide Synthases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007313:Insecta / Q000166:cytology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology
D011993:Recombinant Fusion Proteins; D009243:NAD; D019732:Amide Synthases; C038479:NAD+ synthase