Preliminary Studies on the Expression and Purification of Functionally Active Recombinant Plasmepsin 9 from Plasmodium falciparum

Abstract

Aim: Plasmepsins, a group of homologous aspartic proteinases are attractive drug targets against malaria. Plasmepsin 9 (PM9) expressed in the blood stage of malaria life cycle with unknown function has been strongly considered as a potential target. However, recombinant expression of active PM9 for biochemical and structure-activity analysis of the enzyme has been very challenging. This paper presents preliminary report on the expression and purification of active recombinant plasmepsin 9 from Plasmodium falciparum. Place and Duration of Study: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida between November 2011 and October, 2012. Methodology: The plasmid of truncated proenzyme (proPfPM9) from Plasmodium falciparum was expressed in Escherichia coli Rosetta 2(DE3)pLysS competent cells. The protein was purified from inclusion bodies using combination of cation exchange and gel filtration chromatography. The expression and purification fractions were subjected to SDS-PAGE. The zymogen was activated to produce mature form of active recombinant PfPM9. Catalytic activity test of PfPM9 was determined using a chromogenic substrate, Lys-Pro-Ile-Glu-Phe-Nph*Arg-Leu (RS6). Substrate hydrolysis was examined from substrate cleavage scan.  Results: SDS-PAGE confirmed the expression and purification of truncated proenzyme (proPfPM9) with molecular weight of approximately 54 kD. Kinetic assays showed that the purified PfPM9 was active. The hydrolytic activity of PfPM9 on RS6 was confirmed by a shift in absorbance peak from 280 nm to 272 nm at the end of assay reactions. Inhibition studies on the mature PfPM9 showed that the activity of the enzyme was inhibited by pepstatin A. Conclusion: Protocols for successful expression and purification of active recombinant plasmepsin 9 in E. coli have been established in this preliminary report. These will aid production of sufficient yield of PfPM9 for detailed kinetic and structural characterization of the enzyme and development of future inhibitors against malaria.