The doctoral dissertations of the former Helsinki University of Technology (TKK) and Aalto University Schools of Technology (CHEM, ELEC, ENG, SCI) published in electronic format are available in the electronic publications archive of Aalto University - Aaltodoc.
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Expression, Purification and Characterization of Fungal and Viral Recombinant Proteins

Helena Vihinen

Dissertation for the degree of Doctor of Science in Technology to be presented with due permission of the Department of Chemical Technology, for public examination and debate in Auditorium KE1 at Helsinki University of Technology (Espoo, Finland) on the 22nd of March, 2001, at 12 noon.

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Abstract

This work reports the production of recombinant yeast and viral proteins in a number of diverse in vivo model systems for enzymatic and structural studies. In the first part Hsp150Δ peptide, a derivative of the yeast (Saccharomyces cerevisiae) secretory heat-shock protein Hsp150, was investigated for its ability to act as a carrier in transporting the ectodomain of rat nerve growth factor (NGFRe) out from the yeast cell. The Hsp150Δ-NGFRe fusion protein was efficiently secreted into the growth medium, where it constituted the majority of total secreted proteins. Inhibition experiments with purified Hsp150Δ-NGFRe showed that Hsp150Δ did not prevent NGFRe from folding into a ligand-binding conformation. Circular dichroism (CD) analysis revealed that the Hsp150Δ-carrier did not have any specific secondary structure, which was also suggested by NMR analysis of a synthetic polypeptide corresponding to the repetitive consensus sequence of subunit II of Hsp150. These findings suggest that Hsp150Δ can successfully act as a carrier for foreign proteins, such as NGFRe, made and secreted by S. cerevisiae.

The second part of this study involved the expression and purification of an RNA animal virus, Semliki Forest virus (SFV), nonstructural proteins (Nsp1-4) using a number of in vivo protein expression systems. To ensure quantities large enough for structural and enzymatic studies of the Nsps, each of them was expressed either in bacteria (Escherichia coli) or in insect cells (Sf9). All the proteins were expressed in high quantities (10-100 mg/l), and purified by affinity and size exclusion chromatography under nondenaturing or denaturing conditions. Independent of the expression system used, all the partially purified Nsps aggregated and precipitated either upon concentration, dialysis, storing or thawing. No detergents were found that could alleviate the aggregation problem or assist in the purification process.

Despite the unsuccessful purification of Nsps for structural studies, the expression and partial purification of Nsp1 and Nsp3 permitted biochemical characterization of their enzyme activities and posttranslational modifications. Point mutational analysis of the Nsp1 methyltransferase domain revealed that residue His38 was essential for the guanylyltransferase activity of Nsp1. Furthermore, residues Asp64 and Asp90 were found to be important for the methyltransferase activity of Nsp1. Phosphorylation sites in Nsp3 were determinated by point mutational analysis, electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) as well as by phosphopeptide mapping and Edman sequencing. A phosphorylated domain (aa 320-368) was located in the C-terminal, non-conserved region of Nsp3, where 12 serines and 4 threonines could be modified by phosphates. The phosphorylation of Nsp3 seemed not to affect the membrane association or the localization of Nsp3 in either transfected or infected cells. Furthermore, Nsp3 phosphorylation deficient mutant viruses were capable of replication in infected mammalian cells a similar manner to the wild type SFV, but their neuropathogenicity in adult mice was greatly reduced.

This thesis consists of an overview and of the following 6 publications:

  1. Jämsä, E., Holkeri, H., Vihinen, H., Wikström, M., Simonen, M., Walse, B., Kalkkinen, N., Paakkola, J. and Makarow, M. (1995). Structural features of a polypeptide carrier promoting secretion of a β-lactamase fusion protein in yeast. Yeast 11, 1381-1391. © 1995 John Wiley & Sons Limited. By permission.
  2. Simonen, M., Vihinen, H., Jämsä, E., Arumäe, U., Kalkkinen, N. and Makarow, M. (1996). The hsp150Δ-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae. Yeast 12, 457-466. © 1996 John Wiley & Sons Limited. By permission.
  3. Holkeri, H., Simonen, M., Pummi, T., Vihinen, H. and Makarow, M. (1996). Glycosylation of rat NGF receptor in the yeast Saccharomyces cerevisiae. FEBS Lett. 383, 255-258. © 1996 The FEBS Society. By permission.
  4. Ahola, T., Laakkonen, P., Vihinen, H. and Kääriäinen, L. (1997). Critical Residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities. J. Virol. 71, 392-339. © 1997 American Society for Microbiology. By permission.
  5. Vihinen, H. and Saarinen, J. (2000). Phosphorylation site analysis of Semliki Forest virus nonstructural protein 3. J. Biol. Chem. 275, 27775-27783. © 2000 American Society for Biochemistry & Molecular Biology. By permission.
  6. Vihinen, H., Ahola, T., Tuittila, M., Merits, A. and Kääriäinen, L. (2001). Elimination of phosphorylation sites of Semliki Forest virus replicase protein nsP3. J. Biol. Chem. 276, in press. © 2001 American Society for Biochemistry & Molecular Biology. By permission.

Keywords: yeast, heat-shock protein Hsp150, Semliki Forest virus, non-structural protein, nsp, phosphorylation

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© 2001 Helsinki University of Technology

Last update 2011-05-26