A novel approach for over-expression, characterization, and isotopic enrichment of a homogeneous species of acyl carrier protein from Plasmodium falciparum.
Acyl carrier protein (ACP) plays a central role in fatty acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. Holo-ACP is the obligatory substrate for the synthesis of acyl-ACPs which act as the carrier and donor for various metabolic reactions. Despite its interactions with numerous proteins in the cell, its mode of interaction is poorly understood. Here, we report the over-expression of PfACP in minimal medium solely in its holo form and in high yield. Expression in minimal media provides a means to isotopically label PfACP for high resolution multi-nuclear and multi-dimensional NMR studies. Indeed, the proton-nitrogen correlated NMR spectrum exhibits very high chemical shift dispersion and resolution. We also show that holo-PfACP thus expressed is amenable to acylation reactions using Escherichia coli acyl-ACP synthetase as well as by standard chemical methods.
Sharma
Shailendra Kumar
SK
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Modak
Rahul
R
Sharma
Shilpi
S
Sharma
Alok Kumar
AK
Sarma
Siddhartha P
SP
Surolia
Avadhesha
A
Surolia
Namita
N
D000213:Acyl Carrier Protein / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002247:Carbon Isotopes; D019730:Carbon-Sulfur Ligases / Q000096:biosynthesis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010963:Plasmodium falciparum / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000213:Acyl Carrier Protein; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D006639:Histidine; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase