Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase.

Abstract

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.

• Authors: Marco Bellinzoni, Edda De Rossi, Manuela Branzoni, Anna Milano, Fiorenzo A Peverali, Menico Rizzi, Giovanna Riccardi
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 25 Issue 3 Pg. 547-57 (Aug 2002) ISSN: 1046-5928 [Print] United States
• PMID: 12182838 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Recombinant Fusion Proteins
• NAD
• Amide Synthases
• NAD+ synthase

Topics

• Amide Synthases (chemistry, genetics, isolation & purification, metabolism)
• Amino Acid Sequence
• Animals
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (genetics)
• Insecta (cytology, genetics)
• Kinetics
• Molecular Sequence Data
• Mycobacterium tuberculosis (enzymology, genetics)
• NAD (metabolism)
• Recombinant Fusion Proteins (chemistry, genetics, isolation & purification, metabolism)
• Sequence Alignment
• Sequence Homology