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.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 I
pETK-
kan R
Marker
NTANickelColumnEluate#1
NTANickelColumnEluate#2
SPSepharoseEluate#1
SPSepharoseEluate#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
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
Ligation
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
#29