Linked Life Data

12182838

Source:http://linkedlifedata.com/resource/pubmed/id/12182838

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2002-8-16
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
25
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
547-57
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase.
pubmed:affiliation
Dipartimento di Genetica e Microbiologia, Universitá di Pavia, Via Ferrata 1, 27100 Pavia, Italy. marco.b@ipvgen.unipv.it
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't