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

Abstract

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.

Popular Abstract

Viruses are microscopic organisms that need a host, which usually is a human or an animal, in order to reproduce. The coronavirus is known for causing 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. Dengue virus is also dangerous because in most cases does not show any symptoms and it can even lead to death. Since there is currently no treatment for dengue infection, and due to the fast spreading of coronavirus it is important to understand how these viruses reproduce and to find a way to stop this process.
It is known that proteases are enzymes that are involved in breaking down proteins and helping viruses to replicate. In this paper, 2 such enzymes were produced in a host bacteria called Escherichia coli and then a substrate was used to assess if they are active or not. Their structure was also visualized using specific software such as Chimera and Yasara. The experiments were conducted at Lund University, Kemicentrum, Biotechnology Department.
The Coronavirus outbreak started in Wuhan, China, in December 2019. From there, it spread worldwide from human to human resulting in the covid pandemic declared in March 2020 by the World Health Organization. The origin of the infection and the transmission way is unknown but it is believed to be caused by exposure to infected animals. However, the first coronavirus pandemic was actually registered in 1965, followed by another one in 2003 and 2012. This virus is considered highly infectious due to its rapid spreading and large number of infected cases. Worldwide, there are over 750 million registered cases and almost 7 million deaths.
The dengue virus can cause dengue fever through mosquito bites. It is assumed that it was transmitted from primates to humans approximately 1000 years ago in Africa or Asia. Between 100 and 400 million infected cases of dengue fever are registered yearly in tropical or subtropical regions worldwide but the actual number can be even higher since most of the cases do not show any symptoms.
This research showed that there are quite a few differences between these 2 viruses when it comes to structure and mechanism even though they manifest the same symptoms in infected cases and that coronavirus seems to adapt better to environmental changes than dengue virus. Since the experiments proved challenging with multiple failures and repetitions, more time and research would be necessary for a better understanding of how to stop these viruses from reproducing and spreading further.

• author: Svensson, Raluca
• supervisor: Javier Linares-Pastén
• organization: Biotechnology (MSc); Biotechnology (M.Sc.Eng.)
• course: KBTM01 20232
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: coronavirus, dengue, protease, pandemic, SARS-Cov-2, DENV, viral replication, proteins, Escherichia coli, DL-BAPNA, computational modeling, recombinant protein production, biotechnology
• language: English
• id: 9146174
• date added to LUP: 2024-01-22 11:26:09
• date last changed: 2024-01-22 11:26:09