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Identify Important SUMOylation Site of Influenza Virus Protein Using FRET Technology and New Strategy for Fluorescence Protein Expression

  • Author(s): Jiang, Jin
  • Advisor(s): Liao, Jiayu
  • et al.
Abstract

Influenza, which is also commonly known as "the flu", is an infectious disease caused by an influenza virus. The symptoms of influenza could be mild to severe, including fever, headache, sore throat, feeling tired, headache, coughing, and muscle pains. The influenza virus infects and kills over 100,000 people in the world every year. Due to the fast pace in which influenza virus mutates, the virus itself would develop resistances to the current FDA approved drugs. The influenza non-structural protein (NS1) is related to the SUMOylation pathway, a post-translational modification involved in various cellular processes.

The first part of this thesis is mainly focus on finding out the SUMOylation site of the NS1 influenza protein by using site-directed mutagenesis and Förster Resonance Energy Transfer (FRET) based technology. Since the SUMO protein is conjugated to the lysine residue of the NS1 protein, by mutate each lysine to Alanine and using FRET-based SUMOylation assay to test every mutants of NS1, the Lysine residue K131 was founded to be the SUMOylation site of NS1. This result was further proved by Plaque assay, which showed the mutation of Lysine 131 would affect the viral replication in MDCK cells. Identification of essential SUMOylation site(s) of NS1 can provide supporting evidence to develop new medicine for treating Influenza virus.

The second part of this thesis is focus on designing a new strategy for fluorescence protein expression. Nowadays, using E.coli bacteria as the host for protein production is a very common strategy. However, there are several challenges that needs to be overcome by using E.coli protein production, such as the degradation of the target protein, the toxic of protein towards the host, the misfolding and instability of the protein, and the complex purification procedures. By expression the proteins in inclusion bodies (IBs) would help alleviating these problems. This thesis conducted a signal sequence TMAO-reductase (ssTorA) to drive the target protein Ypet-Uba2, into the inclusion bodies and overcame the degradation of the protein. Also, a buffer screening method was designed to purify the protein from inclusion bodies. As a result, trehalose was proved to be a high efficient additive for the Ypet-Uba2 protein purification.

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