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Optimize Bacterial Protein Expression System for Quantitative FRET Assays of Influenza Virus and Immune Check Point Proteins

  • Author(s): Dong, Xuefeng
  • Advisor(s): Liao, Jiayu
  • et al.
Abstract

Förster resonance energy transfer (FRET) is a technology that analyzes protein-protein interactions using fluorescent proteins tags, such as cyanoprotein (CyPet) and yellow fluorescent protein (YPet). In our lab, we developed a mature quantitative FRET technology to measure protein-protein interactions, especially the interactions of some proteins which be involved in cancer induction and regulation process. Cancer is a disease that when cells and tissues on any part of the body without control. These cells can continue to grow without limitation, invade nearby tissues, and interfere with organ functions. There is one important mechanism involved in cancer induction process which is SUMOylation pathway. SUMOylation pathway has been extensively studied for a long time but the accurate components of SUMOylation are still not very clear. There are lots of induction factors of cancer, one of them has been found recently is influenza virus. Influenza, an infectious disease caused by an influenza virus which can kill many people every year and spread in various methods .It has three different types which are Type A, Type B, and Type C. H1N1 virus is a subtype influenza A virus. It including 8 RNA segments coding for 12 proteins. According to recent reports, some of H1N1 virus proteins can be SUMOylated so that it makes H1N1 has a high investigation value. There are some other proteins can be potential SUMO targets that contribute to cancer induction such as PD-1 and PD-L1.Programmed cell death protein1 (PD-1 or CD279), is known as a very important cell surface receptor of T- cell and plays an important role in the immune reaction mechanisms, such as it can suppress T-cell immune activities. Programmed death-ligand1 (PD-L1 or CD274 or B7-H1) plays an important role in immune system too. In this study, I applied our FRET technology to measure SUMOylation reactions of some H1N1 proteins and do quantitative Kd determination of PD-1/PD-L1. Through with construct design, gene clone, transformation and protein purification to get proteins with high purity. Then I measured Kd and SUMOylation reactions with these proteins. As results, I got the Kd of YPet-NP and CyPet-Ubc9, PD-1 extra-cellular domain and PD-L1 extra-cellular domain. And I found that NP virus protein which is one of H1N1 proteins can be SUMOylated. These results can contribute to protein-protein interaction studies of H1N1 virus and potential drug target detection of PD-1 and PD-L1 pathway.

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