Expression and Purification of the Plasmodium berghei Apical Membrane Antigen-1
1
Production of a Vaccine Candidate: 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 several Plasmodium parasite species, 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 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 the study of possible malaria vaccine candidate proteins. P. berghei Apical Membrane Antigen-1 (PbAMA-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 rodent or other animal model for the initial studies. Results and Discussion Although each step was followed by a DNA gel in the beginning, sent out for sequencing, and the final protein was run on a gel showing the correct molecular weight, a Western blot was also performed using the protein and a positive control. 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. Thankfully, a purification protocol for a 6-His PfAMA-1 had already been developed by the Lanar Lab, which proved to be an effective way of purifying PbAMA-1 as well. Acknowledgements Dr. Evalina Angov provided us with the pETK(-) vector from her work with P. falciparum. Sheetij Dutta for acting as mentor for this project. Lanar Lab for providing materials and other expertise. 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.ht ml Courtesy Dr. Collette Hillier AMA-1 is found on the surface of merozoites throughout the genus Plasmodium. It is considered an erythrocytic stage antigen as it is processed during merozoite invasion of red blood cells. Evidence exists that AMA-1 binds to erythrocytes and may help with parasite invasion. PbAMA-1 gene Not I BamHI 6 His -tag 6507 bp BamHI Not I pETK- kan R Marker NTA Nickel Column Eluate #1 NTA Nickel Column Eluate #2 SP Sepharose Eluate #1 SP Sepharose Eluate #2 Microfluidization Production and Purification of PbAMA-1 Final product Q Ni Creation of Expression Plasmid Figure 1. Digestion of pETK- and PbAMA-1 insert with BamHI and NotI 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. Figure 4: Purification of Final Product 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, which in this case was both of them. The second blot was developed using P. falciparum specific antibodies, which show up on the PfAMA-1 and not PbAMA-1. E. Coli courtesy of http://www.geocities.com/CapeCanaveral/3504/ecgm.jpg 42 kDa 32 19 AMA-1 Conclusions The evidence shows that PbAMA-1 was expressed and purified using the various methods described in this presentation. With all of the experiments showing the correct molecular weights and perfected matched sequence information, it is very unlikely the protein collected could be anything else. Not to mention the quantity of protein produced, far exceeds any other naturally produced protein in the E. coli cells. As of the publishing of this poster the antigen has begun to be used in a mice and we should have results soon. Also, with a human PfAMA-1 vaccine trial coming up in Fall of 2006, the information acquired from this project and those related to it will be very useful. Courtesy of Sheetij Dutta P. Berghei Genomic DNA PCR Amplification Restriction Digest Vector and Insert BamHI Not I in pETK- kan R PbAMA-1 Gene Ligati on pETK- PbAMA-1 Gene Tranformation PCR Colony Screening 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. 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. PbAMA-1 insert pETK - Kanamycin IPTG Bioreactor Flask #2 9
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Transcript of Expression and Purification of the Plasmodium berghei Apical Membrane Antigen-1
Production of a Vaccine Candidate: Expression and Purification of the Plasmodium berghei Apical Membrane Antigen-1
Nathan Jones, Sheetij Dutta, David LanarDivision of Communicable Diseases and Immunology, Department of Immunology
Walter Reed Army Institute for ResearchAbstractMalaria, which is caused by several Plasmodium parasite species, 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
IntroductionPlasmodium 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 the study of possible malaria vaccine candidate proteins. P. berghei Apical Membrane Antigen-1 (PbAMA-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 rodent or other animal model for the initial studies.
Results and DiscussionAlthough each step was followed by a DNA gel in the beginning, sent out for sequencing, and the final protein was run on a gel showing the correct molecular weight, a Western blot was also performed using the protein and a positive control. 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. Thankfully, a purification protocol for a 6-His PfAMA-1 had already been developed by the Lanar Lab, which proved to be an effective way of purifying PbAMA-1 as well.
AcknowledgementsDr. Evalina Angov provided us with the pETK(-) vector from her work with P. falciparum.
Sheetij Dutta for acting as mentor for this project.
Lanar Lab for providing materials and other expertise.
ReferencesSheetij 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 GroupThe Plasmodium berghei research model of malaria
website: http://www.lumc.nl/1040/research/malaria/model.html
Courtesy Dr. Collette Hillier
AMA-1 is found on the surface of merozoites throughout the genus Plasmodium. It is considered an erythrocytic stage antigen as it is processed during merozoite invasion of red blood cells. Evidence exists that AMA-1 binds to erythrocytes and may help with parasite invasion.
PbAMA-1 gene
Not IBamHI 6 His -tag
6507 bp
BamHI
Not IpETK-
kan R
Mar
ker
NTA
Nic
kel C
olum
n E
luat
e #1
NTA
Nic
kel C
olum
n E
luat
e #2
SP
Sep
haro
se E
luat
e #1
SP
Sep
haro
se E
luat
e #2
Microfluidization
Production and Purification of PbAMA-1
Final product
QNi
Creation of Expression Plasmid
Figure 1. Digestion of pETK- and PbAMA-1 insert with BamHI and NotI
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.
Figure 4: Purification of Final Product
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, which in this case was both of them. The second blot was developed using P. falciparum specific antibodies, which show up on the PfAMA-1 and not PbAMA-1.
E. Coli courtesy of http://www.geocities.com/CapeCanaveral/3504/ecgm.jpg
42 kDa
32 19AMA-1
ConclusionsThe evidence shows that PbAMA-1 was expressed and purified using the various methods described in this presentation. With all of the experiments showing the correct molecular weights and perfected matched sequence information, it is very unlikely the protein collected could be anything else. Not to mention the quantity of protein produced, far exceeds any other naturally produced protein in the E. coli cells.
As of the publishing of this poster the antigen has begun to be used in a mice and we should have results soon. Also, with a human PfAMA-1 vaccine trial coming up in Fall of 2006, the information acquired from this project and those related to it will be very useful.
Courtesy of Sheetij Dutta
P. Berghei Genomic DNA
PCR Amplification
Restriction Digest Vector and Insert
BamHI
Not Iin pETK-
kan R
PbAMA-1 Gene
Ligation
pETK- PbAMA-1 Gene
Tranformation
PCR Colony Screening
Figure 2. Shows several PCR screenings of transformedE. Coli colonies. Positives are indicated by a bright band at thecorrect molecular weight and marked with arrows.
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.
PbAMA-1 insert
pETK-
KanamycinIPTG
Bioreactor Flask
#29
