All in vitro platform for rapid protein engineering and analysis based on Leishmania tarentolae

Kovtun, Oleksiy (2012). All in vitro platform for rapid protein engineering and analysis based on Leishmania tarentolae PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Kovtun, Oleksiy
Thesis Title All in vitro platform for rapid protein engineering and analysis based on Leishmania tarentolae
Formatted title All in vitro platform for rapid protein engineering and analysis based on Leishmania tarentolae
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2012-02
Thesis type PhD Thesis
Supervisor Kirill Alexandrov
Robert G. Parton
Total pages 147
Total colour pages 25
Total black and white pages 122
Language eng
Subjects 060112 Structural Biology (incl. Macromolecular Modelling)
060113 Synthetic Biology
060199 Biochemistry and Cell Biology not elsewhere classified
Formatted abstract Cell-free protein translation has recently emerged as a powerful alternative to in vivo-based recombinant protein expression. It enables unmatched approaches to protein engineering, production and labeling. However, the cumbersome preparation of reconstituted translation systems producing correctly folded proteins impedes broad dissemination of cell-free translation technologies. Therefore, the employment of more robust cell-free translation systems and development of connected technologies are highly anticipated.

In the second chapter of my thesis I report the development of a novel translation system based on Leishmania tarentolae (LTE). Unlike other sources for cell-free protein translation systems, this protozoan can be cultivated at high cell density, enabling robust preparation of cellular lysates with high expression activity and full capability for protein folding. Since the Leishmania system was unresponsive to the known cis-elements of RNA messages enhancing translation, a novel efficient 5′ UTR translation enhancer was constructed. In addition to LTE, this artificial sequence is able to promote protein translation in various cell-free translation systems. Therefore 5 this novel enhancer can facilitate the development of new cell-free translation systems by overcoming widespread difficulty with engagement of artificial mRNAs into protein translation.

Availability of highly specific protein binders is essential for the manipulation of target proteins, and is especially important for cell-free systems due to their relatively low protein expression efficiency. In addition, efficient protein purification protocols allow access to multiplexing capabilities of cell-free translation systems. Therefore, in the third chapter of my thesis I report the characterization of a highly specific 15 kDa binder of GFP (GFP-nanobody). Using Xray crystallography and biophysical protein binding analysis we studied structural features resulting in the high affinity and specificity of GFP-nanobody to GFP. Insight into the structure of the complex allowed us to extend the binding specificity of GFP-nanobody to CFP via targeted mutagenesis of this fluorescent protein. We employed GFP-nanobody as a standard tool for manipulation of LTE-expressed proteins.

Using LTE, I developed an all in vitro platform for protein engineering and analysis, which is reported in chapter four of my thesis. Firstly, I devised a modification of PCR, enabling the rapid generation of a library of translation templates, whilst bypassing uneconomical intermediate purification steps. The developed protocol permits streamlined protein engineering and assembly of translation templates using protein-encoding sequences from cDNA libraries, genomic DNA or genetic vectors. Moreover, I have illustrated the utility of this solely in vitro platform for genomewide protein analysis. The platform was further characterized and improved to facilitate the robust preparation of multiple proteins as was shown by isolation of all members of the murine RabGTPase family, many of which are known to resist soluble heterologous expression.

Reconstitution of multisubunit protein complexes often requires coordinated co-expression of several polypeptides, frequently in engineered forms. As a result, many mammalian protein complexes are extremely difficult to reconstitute. Cell-free protein translation systems can readily co-express multiple genes and allow multiplexing of protein translation, thereby facilitating engineering and assembly of multisubunit protein complexes. In chapter five of my thesis I decipher the architecture of the cavin multisubunit complex using the LTE platform, which, until this study, has never been produced in vitro. It has been proposed that the cavin complex contains four related proteins, cavin1-4, and exerts its function in the regulation of caveolae, well-studied plasma membrane invaginations. The LTE-based platform was used to map interactions between full-length and systematically truncated proteins to identify the cavin complex composition and interaction domains within the individual cavins. In particular, all cavins were found to hetero-oligomerize with each other except for the cavin2 - cavin3 pair. Intriguingly, we identified the two subtypes of cavin oligomers, differing by the alternative incorporation of cavin2 or cavin3. Truncation analysis 6 of cavins allowed the identification of a universal interaction domain comprising two conserved coiled-coil regions within primary sequences of all cavins.

In summary, in the course of my project I have developed an all in vitro platform for protein engineering and analysis using a novel cell-free translation system based on Leishmania tarentolae. The platform has been shown to be a robust and efficient tool for different areas of protein research including proteomics, parallelized protein expression and reconstitution of multisubunit protein complexes. Protein products of cell-free translation reactions have been shown to be amenable to single molecule analysis directly in the translation mixture, with no purification required. This facilitates the interrogation of multiple protein parameters across large protein libraries. Using this LTE-based protein production platform, I report the first reconstitution of the cavin complex in vitro. This has allowed mapping of the interactions between individual cavins at high resolution. These results, along with the developed methodology, will now help us to understand the biology of the cavin complex.
Keyword Cell-free translation
Protein expression
Leishmania
Rab GTpase
Cavin
Caveola
Protein complex
GFP binder
Fluorescence correlation spectroscopy
Single molecule detection

 
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Created: Thu, 18 Oct 2012, 16:58:00 EST by Oleksiy Kovtun on behalf of Scholarly Publishing and Digitisation Service