Research toward vaccines against malaria. and in recent years the incidence of malaria has been increasing (5, 25).Vaccination against has the potential to reduce severe malaria-associated morbidity and mortality in areas with the most intense transmission, and it may do so without necessarily preventing blood stage infection (20, 24). Most blood stage vaccine research has been focused on antigens that are expressed on the surface of merozoites (11). Merozoites are released from rupturing red blood cells (RBCs) and quickly invade other RBCs. Merozoite surface protein-specific antibodies, therefore, have only a brief period of time in which they can be active (26). The Xylazine HCl most widely studied merozoite surface protein is merozoite surface protein 1 (MSP1) (15). This molecule is polymorphic and has a complex folding pattern (8, 21). MSP1 Rabbit Polyclonal to XRCC5 is a large (200-kDa) protein. MSP1 is processed into a complex of polypeptides on the merozoite surface, including an 82-kDa N-terminal polypeptide and 30- and 38-kDa central regions, as well as the 42-kDa C-terminal region (MSP142) (1). At the time of RBC invasion, MSP142 is further processed by proteolytic cleavage into a 33-kDa fragment (MSP133), which is shed from the parasite with the rest of the MSP1 complex, and a C-terminal 19-kDa fragment (MSP119). Only the C-terminal MSP119 fragment remains on the merozoite surface and is carried into parasitized RBCs (2). This so-called secondary processing of MSP1 is completed during the successful invasion of a RBC, suggesting that it is a necessary step (3, 7). The MSP119 and MSP142 regions of MSP1 are leading malaria vaccine candidates (15). Studies with rodent malaria and challenge studies with in primates have indicated that vaccines based on MSP119 and MSP142 confer protection against malaria (6, 9, 12, 13, 29, 30). Recently, O’Donnell et al. (22) convincingly demonstrated not only that most sera from two high-transmission areas in Papua New Guinea were able to inhibit parasite invasion in vitro but also that the inhibitory activity was primarily directed against MSP1. By constructing a chimeric transfected line, D-10 (D10-PcMEGF), which expressed an antigenically distinct MSP119 domain from the distantly related rodent species (18, 19), (4), baculovirus-infected insect cells (29), and milk from transgenic mice (30). Recombinant MSP142 produced in baculovirus-infected insect cells (6, 29) and transgenic milk (30) elicits protective responses in an in vivo model system but has yet to be scaled up for human clinical trials. The purpose of the present study was to examine expression for the production of MSP142. The protein expression system, which was the Xylazine HCl first commercialized system for recombinant protein production, is cost-effective and very efficient for nonglycosylated proteins, such as MSP142. MSP1 is a nonglycosylated protein in its native form, and glycosylation blocks the efficacy of MSP142 produced in transgenic milk (30). Here, we describe methods to produce recombinant MSP142 in its correctly folded conformation, to examine the ability of Xylazine HCl antibodies raised against recombinant MSP142 to block erythrocyte invasion by in vitro, and to examine the in vivo efficacy of MSP142 in monkeys against a lethal challenge with MSP142 The amino acid sequence of MSP142 FVO (MSP142 of the Vietnam-Oak Knoll or FVO strain; GenBank accession no. L20092)was used to construct a synthetic gene. The coding sequence of the gene was optimized for expression in by normalizing its AT content on the basis of previously published values for codon bias. This construct, corresponding to amino acids A-1 to S-355, was generated by PCR techniques and was subcloned into a pCR-blunt vector from Invitrogen. The MSP142 FVO gene was then inserted downstream of the T7 promoter by using an expression vector pET24d+ (Novagen Inc., Madison, Wis.) to obtain plasmid pPfMSP142FVOPET. The transcribed sequence of pPfMSP142FVOPET contains an additional LEHHHHHH at the C terminus. BL21(DE3) cells (Novagen) were transformed with pPfMSP142FVOPET and used for expression of recombinant MSP142 FVO protein. Fermentation was performed in a 1.9-liter culture by using defined medium containing (per liter) 13.3 g of KH2PO4, 4.0 g of NH4HPO4, 1.7 g of citric acid monohydrate, 1.2 g of MgSO4 7H2O, 4.5 mg of thiamine-HCl, 25 g of dextrose, 35 mg of kanamycin, and 1 ml of PTM4 trace salts. NH4OH was the nitrogen source, and glucose was the carbon source. Fermentation was carried out at 37C, and once the optical density at 550 nm reached 35, the culture was induced by adding isopropyl-1-thio–galactopyranoside (IPTG) to a final concentration of 1 1 mM. Induction was continued for 3 h before harvesting by centrifugation and cell pellet storage at ?80C. Refolding and purification.