FEBS Letters 352 (1994) 155-158 FEBS 14553 High level expression and characterisation of Plasmepsin II, an aspartic proteinase from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB Plasmodium falciparum Jeffrey Hill”, Lorraine Tyasa, Lowri H. Phylip”, John Kay”, Ben M. Dunnb, Colin Berrya “Department of Biochemistry, University of Wales College of Cardijjf PO Box 903, Cardiff CFI 1Sr Wales, UK bDepartment of Biochemistry and Molecular Biology, J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610, USA Received 22 July 1994 Abstract DNA encoding the last 48 residues of the propart and the whole mature sequence of Plasmepsin II was inserted into the T7 dependent vector PET 3a for expression in E. coli. The resultant product was insoluble but accumulated at -20 mg/l of cell culture. Following solubilisation with urea, the zymogen was refolded and, after purification by ion-exchange chromatography, was autoactivated to generate mature Plasmepsin II. The ability of this enzyme to hydrolyse several chromogenic peptide substrates was examined; despite an overall identity of -35% to human renin, Plasmepsin II was not inhibited significantly by renin inhibitors. Key words: Aspartic proteinase; Plasmepsin II; Plasmodium falciparum 1. Introduction During the blood borne stages of human infection by the malarial parasite Plasmodium falciparum, haemoglobin is di- gested in huge amounts as a source of nutrients to support metabolism. These reactions occur in a specialised, lysosome- like organelle, the digestive vacuole. Aspartic proteinase in- volvement in this processing has been established by de- monstrating that the first step in haemoglobin degradation is inhibited by pepstatin [l]. To date, two aspartic proteinases have been isolated from parasite food vacuoles [2] and the genes encoding both enzymes have been cloned and sequenced [3,4]. These enzymes have now been systematically renamed Plasmepsin I (replacing previous names Aspartic haemoglobi- nase I [2] and Plasmodium aspartic proteinase G [4]), and Plasmepsin II (replacing Aspartic haemoglobinase II [2] and Plasmodium aspartic proteinase D [4]). The Plasmepsin pro- teinases (EC3.4.23.38) thus represent important new targets for antimalarial chemotherapy. Elucidation of structure/activity relationships for both enzymes would facilitate the develop- ment of potent, specific inhibitors for potential application as drugs. Since only trivial amounts of material can be isolated directly from the parasites, production of each enzyme as a recombinant protein is essential. To this end, this report de- scribes the production of Plasmepsin II in E. coli in sufficiently high amounts to permit structural studies to be initiated to- gether with initial characterisation of the recombinant enzyme. high levels of expression of recombinant proteins as fusions with a 13 amino acid leader sequence derived from the N-terminus of the T7 gene 10 protein [5]. The sequence of a positive clone containing the Plasmep- sin II insert in the correct orientation was determined in its entirety to ensure that no mutations had been introduced during the polymerase chain reaction. 2.2. Induction E. coli BLZl(DE3)pLysS cells transformed with the recombinant plasmid were grown to an Am of 0.4 in LB medium supplemented with 150 &/ml ampicillin, and were induced by the addition of isopropyl-b- o-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. Incubation was continued at 37“C for a further 2 h, at which time the cells were harvested by centrifugation (3,000 x g for 10 min) and resus- pended in TN buffer (50 mM Tris-HCl, pH 7.2, 0.15 M NaCl). Ly- sozyme was added (final concentration = 10 @ml) and the cells were lysed by freezing/thawing. 2.3. Purification and refolding 2. Materials and methods 2.1. Cloning The gene encoding the last 48 residues of the pro region and the whole of the mature Plasmepsin II was amplified from the pBluescript KS+ clone 0075M [4] in a polymerase chain reaction using synthetic primers S-CCGGAATTCGGATCCGAACATTTAACTATTGG-3’ and the M 13 universal primer, 5’-GTAAAACGACGGCCAGT-3’. Following digestion of the amplified product with BamHI, fragments were ligated into pET3a (AMS Biotechnology, Milton Keynes, Bucks., UK) that had been cut with BamHI and phosphatase-treated. This vector permits Lysed cells (from 1 1 of culture) were diluted to 200 ml in TN buffer and stirred overnight at 4°C. Insoluble material containing the recombinant protein was pelleted by centrifugation at 16,000 x ,g for 30 min. The pellet was resuspended in 200 ml buffer B (0.1 M Tris-HCl, pH 11 .O, 50 mM j?-mercaptoethanol) and stirred at 4°C for 4 h. Insol- uble material was re-pelleted and washed with a further 200 ml buffer B. After centrifugation, the washed pellet was resuspended in 10 ml buffer C (6 M urea. 0.1 M Tris-HCI. DH 8.0. 1 mM elvcine. 1 mM EDTA, 50 mM /?-mercaptoethanol) add stirred ovemKht at’4”C to solubilise the recombinant material. Residual insoluble material was removed by centrifugation at 28,0000x g for 2 h. The supematant containing recombinant protein was rapidly diluted into 2 1 buffer D (10 mM Tris-HCl, pH 8.5) and stirred at 25°C for a further 24 h to allow refolding of the recombinant protein. This solution was concen- trated to a volume of 30 ml using a Filtron Ultrasette, 5 kDa cut-off tangential flow concentrator (Flowgen Instruments Ltd, Sittingboume, Kent, UK) and the concentrate was loaded onto DEAE-cellulose Pro- ductiv PSClO-DE column (BPS Separations Ltd., Spennymoor, Co. Durham, UK) equilibrated in 0.1 M Tris-HCl buffer, pH 8.5. After extensive washing, the recombinant protein was eluted by a linear gradient (50 ml each) of O-0.8 M NaCl in the same buffer. 2.4. Analytical measurements *Corresponding author. Fax: (44) (222) 874116. SDS-PAGE was performed by the method of Laemmli [6] and gels were stained with Coomassie blue. Edman degradation was performed after blotting samples onto polyvinylidene difluoride (PVDF) as de- scribed previously [4]. Derivation of kinetic parameters (K,,,, k,,,) for the hydrolysis of synthetic chromogenic substrates and K, values for the interaction of naturally-occurring and synthetic inhibitors was carried out as described previously [7j. 0014-5793/94/$7.00 0 1994 Federation of European Biochemical Societies. All rights reserved. SSDI 0014-5793(94)00940-6