Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase.
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 (approximately 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at approximately 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 approximately 100 kDa, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of approximately 30 U.mg(-1) and K(m2PGA) = 0.041 +/- 0.004 mm. The Michaelis constant for the reverse reaction (K(mPEP)) is 0.25 +/- 0.03 mm. pH-dependent activity measurements gave a maximum at pH 7.4-7.6 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 0.18 +/-0.02 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.
Pal-Bhowmick
Ipsita
I
Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
Sadagopan
K
K
Vora
Hardeep K
HK
Sehgal
Alfica
A
Sharma
Shobhona
S
Jarori
Gotam K
GK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007106:Immune Sera; D007700:Kinetics; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D007106:Immune Sera; D010751:Phosphopyruvate Hydratase