Expression in E. coli and purification of the nucleoside diphosphate kinase b from Leishmania major.
Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDK) are involved in the salvage pathway by which free purines are converted to nucleosides and subsequently to nucleotides. In this report, we describe the cloning of NDK coding-sequence from Leishmania major, the expression of the enzyme containing a His(6)-tag in Escherichia coli, and purification of the catalytically active native protein by affinity chromatography using Ni-NTA resin.
de Oliveira
Arthur H C
AH
Departamento de Química, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto-SP, Brazil.
Ruiz
Jerônimo C
JC
Cruz
Angela K
AK
Greene
Lewis J
LJ
Rosa
José C
JC
Ward
Richard J
RJ
D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D016773:Leishmaniasis, Cutaneous / Q000201:enzymology / Q000453:epidemiology / Q000235:genetics; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*
D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D009701:Nucleoside-Diphosphate Kinase