Website Text
Work at UMBI
Research focus: To use aparasite, Perkinsus marinus, based expression system to produce recombinant proteins for Malaria with the ultimate endpoint being the characterization of the recombinant proteins and the production of Malaria Vaccines
I most recently served as a postdoctoral research fellow at the University of MD Biotechnology Institute(1) in the laboratory of Jose Fernandez Robledo(2) and Gerardo Vasta (3) My research work involved the design and engineering of DNA vectors expressing either red tomato (RT)fluorescent protein or green fluorescent protein(GFP) driven by the promoter of a highly expressed protein of Perkinsus marinus (4)the parasite organism used for production of recombinant proteins. Perkinsus marinus (P. marinus) is the parasite responsible for the oyster disease, ‘Dermo’ (5) which has been responsible for much of the oyster mortality of natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States since the 1950’s.
P. marinus is an ideal system for the production of recombinant proteins for four reasons:
Malaria (7) is a mosquito -borne infectious disease of humans. It is found primarily in the subtropical and tropical regions of Sub-Saharan Africa, Asia and the Americas. Malaria is most prevalent in these areas because of the high amount of rainfall and consistently high temperatures which provide an environment ripe for perpetual breeding of mosquitoes. Malaria claims the lives of more children worldwide than any other infectious disease and over 1 million people die each year. Malaria is a major public health problem (8).which affects over 100 countries and over 40% of the world’s population. The development of Malaria vaccines is a research area of intensive work. The efficacy of malaria vaccines, however, has been confounded by the development of drug resistant parasites and insecticide resistant mosquitoes.
The etiological agent responsible for the most severe form of malaria is the apicomplexan(9) parasite Plasmodium falciparum. (10)The parasite undergoes a very complex life cycle with 2 stages; a sporocytic replication in the salivary gland of the female Anopheline mosquito (11) and schizogonic replication in the liver and erythrocytes of a human host. When the carrier mosquito bites a human, a small quantity of mosquito saliva is injected along with 5-200 P.falciparum sporozoites (12). The sporozoites move through human bloodstream and are able to infect human liver cells within minutes. In the liver, the parasite undergoes replication and reinfection with rupture of some of the infected Human liver cells. After release from the liver, the p.falciparim merozites(13) move into the blood stream to infect circulating erythrocytes. The precise mechanism of invasion has not been elucidated, however, it appears utilize proteins present in the apical complex (14). The apical complex, comprised of proteases and other proteins, is believed to be involved in binding and invasion of erythrocytes. Apical membrane antigen 1 (Ama1)(15) appears to be a critical P. falciparum binding protein involved in the invasion of cells. Ama1 initially binds an erythrocyte binding protein and then binds glycophoinA (16)], an erythrocyte cell surface protein. MSP1 (17), a merozite surface protein, binds heparin-like molecules. A set of proteases degrade a portion the erythrocyte cells hemoglobin which is used by the parasite for de novo synthesis of protein. After invasion, the parasite loses its apical complex, dedifferentiates into a trophozoite (18)] and is found in a vacuole within the red blood cell cytoplasm. The trophozoites grow and then undergo a schizogonic division which releases more merozoites to infect more erythrocytes.
We focused on producing recombinant proteins thought to be involved in binding and invasion: Ama1, and the serine repeat antigens, Sera 4S and Sera 5S. The genes were PCR amplified, ligated to a TA cloning vector and transformed into JM109 bacteria. The transformed bacteria were grown overnight and plasmid DNA was Isolated. Plasmid DNA was initially sequenced using T7 and Sp6 primers. If the gene of interest was large, internal primers were designed and used for sequencing. All sequencing data was aligned using Sequencher. Silent mutations and other changes were noted and the best clone was chosen for cleavage and ligation into our RT or GFP expression vector. The final recombinant DNA constructs contained the recombinant gene of interest, the fluorescent protein and a histidine tag. This DNA construct introduced into Perkinsus marinus using electroporation. Expression of the recombinant protein was monitored by fluorescence microscopy. Not all cells contained the recombinant plasmid. Approximately 2 out of every 500 (0.4%) cells scored were producing GFP or RT. This mixed population was grown to high density for isolation of protein and purification using a nickel based columns. Recombinant protein was recovered from only 1.2 x 107 cells expressing GFP.
Links and Citations:
Page last updated: July 26, 2011 16:43