Inari Kursula research group is looking for a student to a project dealing with preparation of N-terminally acetylated PfActI for structural and biochemical studies.
The aim of the project is to generate a native N-terminus on our currently functional recombinantly expressed PfActI construct that can be purified in significant quantities for biochemical, biophysical and structural studies and to characterize the protein by several biochemical assays that we have established for our current construct.
Actin is a cytoskeletal protein that forms thin filaments within cells and has a plethora of functions (muscle contraction, cell shape maintenance etc.). Our group focuses on the study of actin and actin-binding proteins in Plasmodium spp., the causative agent of malaria, where actin specifically has diverged evolutionarily from the so-called canonical eukaryotes. In Plasmodium, the major actin isoform, actin I (PfActI), is constitutively expressed throughout the life cycle of the parasite and has roles in invasion of and egress from red blood cells as well as in gliding motility, the active mechanism whereby the parasite moves within its hosts and across biological barriers.
We express PfActI recombinantly in insect cells using baculoviral techniques. Our current constructs result in a non-native N-terminus. In mammalian actins, the processing of the N-terminus is crucial for actin-myosin binding as well as other filament binding interactions. Previous work has shown that the N-terminus of PfActI derived from the parasite is acetylated. The master's project consists of (1) the preparation of a suitable construct for expression and purification of PfActI with a native N-terminus and (2) characterization of the differences the old and the new constructs might have in terms of polymerization, ATP hydrolysis and myosin binding. The exact details of the analyses will be decided together with the student.
Experience in recombinant protein expression and purification is desired. Further, interest in cytoskeletal proteins, infection biology and protein chemistry are beneficial. The project will contain the use of at least the following techniques: recombinant insect cell expression, SDS-PAGE, IMAC, general protein purification techniques, mutagenesis, fluorescence-based measurements of enzymatic activity and electron microscopy.
Interested? Send an email with CV and a transcript of records to Esa-Pekka Kumpula and Inari Kursula (firstname.lastname (at) oulu.fi).
Esa-Pekka Kumpula PhD Student Group of Prof. Inari Kursula
Faculty of Biochemistry and Molecular Medicine (FBMM) PO Box 3000 FI-90014 University of Oulu FINLAND
Office: F222 Lab: F221