The production of recombinant 6LZE_A and NS2B-NS3 in E. coli

Detta är en Master-uppsats från Lunds universitet/Bioteknik (master); Lunds universitet/Bioteknik (CI)

Sammanfattning: Even though the first coronavirus pandemic was registered in 1965, SARS-CoV-2 was better acknowledged when it caused the worldwide pandemic in 2019 in which millions of individuals lost their lives. Due to its facile way of spreading from one individual to another, the number of infected cases has gone up to more than 750 million to this day. DENV is a very dangerous virus due to its asymptomatic profile in most of the infected cases. If left untreated it can lead to dengue hemorrhagic fever and for individuals who get infected with the virus for the second time, the consequences can be lethal. Since there is currently no treatment for dengue infection, and due to the fast spreading of SARS-CoV-2 that still leads to deaths every day, it is important to understand how proteases are involved in viral replication and thus to find a way to stop this process in order to avoid spreading these viruses further. In this report, 2 proteins (6LZE_A and NS2B-NS3) from these viruses were produced in Escherichia coli and their biological activity has been tested on DL-BAPNA substrate. Both of these proteins are structures of the main proteases that cleave the polyproteins, this step being very important in the replication process. Furthermore, computational modeling was used to understand the differences between their structures and how their catalytic mechanisms work. The results showed that working with these proteins has been proven difficult at times due to several failures and repetitions either because very few colonies grew on the Agar plates, cells died during the cultivation process, low concentrations, or failed purification. Hence, more time and research would be necessary to fully understand the reasons behind these issues. However, this report managed to highlight the different production times between the proteins and how SARS-CoV-2 seems to have better environmental stability than DENV due to its structure and docking characteristics.

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