Expression and Purification of the Plasmodium berghei Apical Membrane Antigen-1 Nathan Jones, Sheetij Dutta, David Lanar Division of Communicable Diseases and Immunology, Department of Immunology Walter Reed Army Institute for Research Abstract Malaria, which is caused by Plasmodium parasite, is estimated to be responsible for 2.7 million deaths each year. Apical Membrane Antigen-1 (AMA-1) is an important vaccine candidate for malaria. It is present on the surface of invading merozoite and sporozoite stages of the parasite and mediates invasion into both red blood cells and liver cells. Antibodies to AMA-1 inhibit these invasion events; hence, there exists a strong rationale for a future malaria vaccine based on AMA-1. To test this rationale we would like to set-up a rodent model to study AMA-1 immunogenicity. This model requires the expression and purification of a rodent malaria parasite protein P. berghei AMA-1 (PbAMA-1) in an Escherichia coli host. My project included designing polymerase chain reaction (PCR) strategies to amplify the AMA-1 gene using P. berghei genomic DNA; cloning this gene into an expression vector, derived from pET32; and transformation of the recombinant plasmid into an expression host, E. coli Tuner strain. This was followed by small scale and large scale expression cultures. The PbAMA-1 protein was purified using a two step chromatographic procedure, then characterized by SDS-PAGE and Western Blot. The final product is currently being used as a vaccine in mice and its ability to protect against live blood stage and sporozoite stage challenge is being assessed   Results and Discussion All steps of cloning were folowed by DNA gel examination. The ~1.4 kb gene was PCR amplified and cloned into pETK- vector. Sequencing, showed the correct gene was cloned. The PbAMA-1 protein was expressed by the E. coli as evidenced by westerb blotting with a 6His monoclonal antibody. On a gel this protein had the correct molecular weight of about 54 kDa. One of the key events in the whole process was the primer design, which contained the information for the restriction enzyme sites an for a 6-Histidine tag. These modifications made identification and purification several fold easier. A purification protocol used for PfAMA-1 purification was also applied purify PbAMA-1 to homogeneity. Creation of Expression Plasmid pETK- PbAMA-1 Gene BamHI P. Berghei Genomic DNA Not I pETK- 6507 bp kan R Figure 1. Digestion of pETK- and PbAMA-1 insert with BamHI and NotI Figure 5. Shows two Western blots each with a lane of a P. falciparum AMA-1 protein and a lane with PbAMA-1. The first blot was developed with antibodies for any protein containing a 6-Histidine tag, hence both protein bands . The second blot was developed using P. falciparum specific antibodies,hence, the PbAMA-1 band did not show up. PCR Amplification Restriction Digest Vector and Insert PbAMA-1 gene Not I BamHI 6 His -tag Ligation Figure 2. Shows several PCR screenings of transformed E. Coli colonies. Positives are indicated by a bright band at the correct molecular weight and marked with arrows. BamHI Not I in pETK- kan R Introduction Plasmodium berghei is one of four malaria species that infect murine rodents in Africa. Although the infection of rodents has no major effect on people, even those who live in these areas, rodent species are particularly suited for studies relating back to humans. The life cycle of rodent malaria, as well as the parasites morphology, exactly mirrors that of human malaria. Also, on a more subtle level genomic organization, parasite metabolic pathways, various attachment proteins, and drug sensitivity and resistance have all shown to be conserved between rodent and human parasites. However, there are minor differences between the parasites such as overall size and certain host specific adaptations. This project was mostly concerned with expression of the P. berghei Apical Membrane Antigen-1 (PbAMA-1). AMA-1 has been shown to exist on the surface of the merozoite stage of the parasites. It has also been shown to that antibodies to its P. falciparum homolog can inhibit in vitro erythrocyte invasion by the parasites. This inhibition indicates that P. falciparum AMA-1 might be a good vaccine candidate. In order to study the potential vaccine candidate’s efficacy in vivo it is necessary to use a an animal model for initial studies. Conclusions The evidence shows that PbAMA-1 was expressed and purified to homogeniety using the methods described in this presentation. With all of the experiments showing the correct molecular weights and sequence, the protein made during this project is now being used in a mouse vaccine trial and we should have results soon. Also, with a human PfAMA-1 vaccine trial coming up in Fall of 2006, the information acquired these trials will determine if AMA-1 is a good vaccine candidate for malaria. PbAMA-1 Gene Tranformation #29 pETK- PCR Colony Screening PbAMA-1 insert Figure 3. Following the PCR screen the colonies who were shown positive were grown in small cultures, mini-prepped, and digested to be sure they were positive. Also, #29 was sent out for sequencing and was positive. E. Coli courtesy of http://www.geocities.com/CapeCanaveral/3504/ecgm.jpg The expression plasmid was heat shocked into competent XL1 Blue and Top10 E. coli, then plated on L.B. Agar with kanamycin. Colonies were chosen at random from the plates and screened using PCR. Acknowledgements Sheetij Dutta for acting as mentor for this project. Dr. Lanar Lab for providing materials and other expertise. Evelina Angov provided us with the pETK(-) vector. Col Jack Williams provided us the P. berghei parasites. Column1 Eluate #1 Column 1 Eluate #2 Marker Tuner Expression Host Col 2 Eluate #1 Col 2 Eluate #2 Figure 4: Purification of Final Product Courtesy of Sheetij Dutta 42 kDa 32 19 AMA-1 Production and Purification of PbAMA-1 Courtesy Dr. Collette Hillier AMA-1 is found at the apical end of invading merozoites throughout the genus Plasmodium. The AMA-1 gene cannot be knocked out hence, it is critical for host cell invasion and parasite survival. Evidence exists that AMA-1 binds erythrocytes and allows reorientation of the merozoites during invasion. References Sheetij Dutta, J. David Haynes, Arnoldo Barbosa, Lisa A. Ware, Jeffrey D. Snavely, J. Kathleen Moch, Alan W. Thomas, and David E. Lanar. Mode of Action of Invasion-Inhibitory Antibodies Directed against Apical Membrane Antigen 1 of Plasmodium falciparum Infection And Immunity. 2005 April; 73(4):2116–2122. The Leiden Malaria Research Group The Plasmodium berghei research model of malaria website: http://www.lumc.nl/1040/research/malaria/model.html Microfluidization Final product 2 1 Kanamycin IPTG Bioreactor Flask