tailieunhanh - Báo cáo khoa học: Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase

We have cloned, over-expressed and purified enolase from Plasmodium falciparumstrain NF54 in Escherichia coliin active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequen-cing. The recombinant enolase (r-Pfen) could be obtained in large quantities ( 50 mg per litre of culture) in a highly purified form ( 95%). The purified protein gave a single band at 50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean ± SD mass of 51396 ± 16 Da, which is in good agreement with themass calculated fromthe sequence | Eur. J. Biochem. 271 4845-4854 2004 FEBS 2004 doi Cloning over-expression purification and characterization of Plasmodium falciparum enolase Ipsita Pal-Bhowmick K. Sadagopan Hardeep K. Vora Alfica Sehgal Shobhona Sharma and Gotam K. Jarori Department of Biological Sciences Tata Institute of Fundamental Research Mumbai India We have cloned over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase r-Pfen could be obtained in large quantities w 50 mg per litre of culture in a highly purified form 95 . The purified protein gave a single band at 50 kDa on SDS pAgE. mAlDI-TOF analysis gave a mean SD mass of 51396 16 Da which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was w 100 kDa indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of 30 U-mg 1 and Km2PGA mM. The Michaelis constant for the reverse reaction KmPEP is mM. pH-dependent activity measurements gave a maximum at pH irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na whereas K has a slight activating effect. The cofactor Mg2 has an apparent activation constant of O. 02 mM. However at higher concentrations it has an inhibitory effect. Polyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite Plasmodium yoelii which shares 90 identity with the P. falciparum protein. Keywords enolase homodimer localization Plasmodium falciparum purification. Malaria remains

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