| |
||
Joshua
Zhang '07, undergraduate research fellow with
Dr. Alan Cowman,
HHMI International Research Scholar
Research Poster
|
|
Zhang recounts his experience:
This summer, I worked in the lab of Dr. Alan Cowman at the Walter Eliza Hall Institute for Medical Research in Melbourne, Australia, investigating proteins involved in the invasion process of the Malaria parasite Plasmodium falciparum into red blood cells. Dr. Cowman is best known for being one of the first researchers to transfect genes into P. falciparum around ten years ago, and to this day his lab is one of the most distinguished labs in the world on malaria research. Researchers from around the world come to his lab to learn how to transfect into malaria parasites. Working in a world famous research institute was an amazing experience; different from the research I have conducted here at Kenyon, and I was delighted to have the opportunity to study outside of the United States.
Melbourne was a wonderfully modern city located in southeast Australia, and is the second largest city in Australia behind Sydney, with a population of over 3.6 million. Although it was winter my entire stay, the weather was very mild, with average temperatures of 45-55 oF. As I found out, Australia is a sports nation, and Melbourne is the sports capital of Australia. My research opportunity was timed perfectly with the world cup, and every game I joined tens of thousands of Socceroo fans on Federation Square to watch the games. Over fifty thousand people watched the last game of the Socceroos, and even in defeat the Australians were proud of their team. Melbourne’s sports atmosphere was unlike any I had ever seen.
The international culture was also fantastic. Melbourne has a very large Greek population and Jewish population. International scholars from all over the world came and went, and very few Australians born residents lived at the Graduate House, where I stayed during my time in Melbourne. Different cuisines were abound, with Italian restaurants lined up on Lygon street, Asian shopping in the Chinatown on Little Burke street, and a variety of other cultural fascinations within a twenty minute tram ride from the Graduate House. Immediately outside Melbourne proper, within a day’s car ride, I was able to see Koalas, Penguins, Kangaroos, Wombats, and a variety of other animals native to Australia, on Phillip Island, the Great Ocean Road, and the mysterious Mount Dandenong.
|
Zhang spent many hours in the lab--and
many hours on weekends touring the sights of Melbourne.
|
I spent an average of about nine hours in lab every week day, and about an hour on the weekends. The Cowman Lab consisted of eleven post-doctoral students, and only one Ph.D student. The unusual post-doc to grad student ratio was due to the prestige of the lab. Being one of the foremost labs in malaria research gave Dr. Cowman an impressive influx of post-doc students who were interested in his lab! I worked under the guidance of Dr. Jake Baum, a post-doc in his third year in the Cowman lab from London. Each of the scientists in the lab worked on a different aspect of malaria, from the reproduction cycle within the mosquito to the invasion process of the malaria parasite to the reuptake of the parasite by another mosquito. My project dealt with the actin-myosin motor that propels the parasite into red blood cells, specifically the protein CAP (adenylate Cyclase Associated Protein). It was important to study CAP, since CAP is believed to have a role in actin polymerization/depolymerization. Since CAP affects actin, actin powers the actin-myosin motor, the motor powers invasion, and invasion of malaria underlies disease, understanding CAP’s function may lead to a possible pathway of disease prevention.
Plasmodium falciparum is a protozoan parasite responsible for the most dangerous of malaria infections. It is transmitted by the Anopheles mosquito, and causes more than three-fourths of all malaria infections in the world. Once the malaria is transmitted into the blood by the mosquito, the parent form, called a sporozoite, attacks liver cells. After the liver cell has been successfully invaded, the cell bursts to form merozoites, which invade red blood cells. The parasite goes through a 48-hour invasion process, separated into the ring, trophozoite, and schizont stages, ending in the lysing of the cell and the release of 16 to 32 daughter merozoites into the bloodstream, which further invade other red blood cells.
My research was centered on two main projects: the expression of recombinant HIS/GST tagged CAP, and the transfection of CAP tagged with a GFP marker into P. falciparum. HIS is a 6-histidine residue tag, GST (Glutathione S-transferase) is a tag used in expression and purification of a protein, and GFP (green fluorescence protein) is a tag that will fluoresce to show the location of a protein.
My first project was to express recombinant HIS/GST tagged CAP. The PfCAP was obtained by PCR in cDNA (PfCAP has one intron). The His-tagged version of CAP was then cloned into a pProExHTb vector, and the GST-tagged CAP was cloned into a pGEX4T vector. Both of these were then retransformed into E. coli BL21 cells.
The next step was to perform a large scale N’ terminus tagged protein purification protocol. Bacteria was grown and induced with IPTG (isopropyl β-D-thiogalactopyranoside). The cells were then lysed, solubilized in phosphate buffer, and sonicated. The soluble portion of the supernatant was then passed over agarose column, and an optical density was recorded and the highest concentrated fractions were pooled together and run under a protein gel. The results of the protein gel for His-CAP are shown:
The His-CAP gel contained fractions 4, 5, and 6 of the agarose column with a reducing agent, with 5 being the most concentrated fraction. The second gel contained the same fractions without reducing agent. Clearly, our desired His tagged CAP was present, as indicated in the figure (His-CAP has a MW of 21.8 kDa, which is the approximate size of the band). There also appears to be dimerization, as indicated by the second band. The interesting smearing at the top of each fraction may be evidence supporting CAP forming massive conglomerations.
Next, the results of the protein gel for GST-CAP are shown. For GST-CAP, the first gel contained fractions 2, 3, and 4 of the agarose column without reducing agent, with 3 being the most concentrated. The second contained the same fractions with reducing agent and boiled at 100oC briefly. The third gel contained the same fractions with reducing agent at room temperature. All three gels show once again that our desired product, GST-CAP, was present (GST-CAP has MW of 45.7 kDa, which appears as a band in the middle of the fractions). Once again, the smearing at the top suggests a tendency for CAP to form conglomerations of protein complexes.
My second project involved transfection of CAP tagged with GFP into P. falciparum. The GFP is tagged to the C’terminus of CAP. The CAP-GFP is episomal (not integrated into the genome), and is under the AMA1 promoter (in the late schizont stage of the merozoite). CAP was cloned into a pENTR_1,2 Gateway vector. Using LR Multi-site Gateway technique, a pAMA1_CAP_GFP clone was formed. This was then transfected into 3D7 P. falciparum, and no signs of parasites were present upon my departure of WEHI (was on day 17, typical signs of parasites first come in around 28-36 days). I also attempted to tag CAP with an HA tag, but no positive clones were found when cultured.
Once again, as the protein gels seem to show, CAP is definitely expressed, but did not seem to be monomeric. Many other attempts to break up the complexes were attempted (running IPTG at 30oC instead of 37oC, added Urea, etc) but obtained similar results of polymerization. CAP was then sent off to Bondura to have antibodies in rabbits made. This will then make western blots possible, as well as other techniques to see what binds to CAP. There were many future ideas I would have loved to continue on but did not have time for. Some of these included using the anti-CAP antibodies for localization, expression, and immunoprecipitation. Other possibilities involve passing parasite material over a His or GST-CAP column and see what binds to it (possibly Actin). It would have been particularly interesting to see In vitro what effect CAP has on Actin dynamics. Finally, I would have also liked to have used the CAP-GFP for localization and co-localization with other actin-regulating components. I did not get to use the CAP-GFP after successfully making it, but Dr. Baum and the Cowman Lab promised to make good use of it in future studies of malaria invasion!
Australia was an amazing experience both academically and socially. The sports mad nation was a phenomenon I had never really experienced in the USA (not even superbowl weekend!!) and the emphasis on lab work and science had minute differences that added up to the perfect summer research project. Through the interesting accent differences, the “culture shock” that I went through in the first few days of being in Melbourne, and the bountiful variation of Australian fauna, I obtained a well-rounded summer of learning! I would like to one day return to Melbourne with my fellow HHMI research mates, and do graduate or post doctoral work in the field of parasitology. I am very grateful to the Howard Hughs Medical Institute for providing the generous grant to be able to go to Australia, and Kenyon College for providing me with all the necessary knowledge and tools for being a successful researcher. |
