Synthetic proteins can be created by remodelling existing native proteins or by a de novo protein synthesis. Because of their characteristics, these proteins are frequently used in many biotechnological and therapeutical applications. Designer proteins with specific three-dimensional structure and function may be useful as modulators of the immune system, as self-assembling biomaterials or as sensors in different biotechnological systems. Furthermore, the process of artificial protein modelling and synthesis allows us to better understand the basic principles of protein folding and also the relationship between three-dimensional protein structure and protein function. Pizza α helices is a synthetic protein composed of one-third of Pizza2-SR protein with β propeller fold and a two-helix bundle from DF1-L13A protein. Based on the predicted structure of Pizza α helices, we anticipate a spontaneous intermolecular interaction between Pizza α helices protein monomers, which consequently leads to the formation of a self assembling hexamer complex in the shape of a triangular prism named Pizza α helices protein cage. Individual monomers interact through trimerisation of protein region with β-propeller fold and also through antiparallel dimerisation of two two helix bundles in the presence of divalent metal ions. The purpose of my work was to prepare a plasmid construct with a correct nucleotide sequence of Pizza α helices synthetic protein, to express the designed protein in expression bacterial strain and finally to isolate the protein from bacterial cells. Using polymerase chain reaction (PCR), we prepared two different Pizza α helices constructs (Pizza-2hx-AM and Pizza-2hx-bM), which were extracted from bacterial cultures by immobilised metal affinity chromatography (IMAC). After that, using size-exclusion chromatography (SEC), we proved the presence of an equilibrium between different Pizza-2hx-AM oligomeric complexes in distinct buffer solutions. We could determine that the highest quantity of Pizza α helices hexamer was detected in a buffer solution containing a low concentration of a divalent metal ion chelator EDTA. These findings were additionally confirmed with native polyacrylamide gel electrophoresis (Native PAGE), glutaraldehyde crosslinking followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by determination of the molecular weight of protein complexes using multi-detector system. Using SEC, we have also determined that protein construct Pizza-2hx-bM does not form a hexamer complex. Finally, we wanted to confirm the predicted three dimensional structure of the Pizza α helices hexamer complex using X-ray crystallography. Unfortunately, in the protein crystallisation experiment, we were not able to grow protein crystals, which would be adept for X-ray analysis.
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