Skip to main content

LUP Student Papers

LUND UNIVERSITY LIBRARIES

Production of recombinant NS2B-NS3 in E. coli and inhibition studies

Hallberg, Isak LU (2024) KBTM05 20241
Biotechnology
Biotechnology (MSc)
Abstract
The recent 2019 COVID pandemic elucidated the tremendous effects a viral outbreak can have on the societies of the world with almost 7 million cumulative deaths worldwide. It is therefore of high importance to prepare preventative measures to aid the handling of emerging viral threats before a new pandemic is realized. One of the more prevalent and rapidly spreading viruses today are the Dengue virus (DENV). When looking closer at the viral cascade involved in infecting the host, one enzyme plays an especially important role - the NS2B-NS3 protease. Previous endeavors in the field of bioinformatics proved that the addition of a histidine-tag, that is used for purification, may be problematic in the case of the NS2B-NS3, as the tag seem to... (More)
The recent 2019 COVID pandemic elucidated the tremendous effects a viral outbreak can have on the societies of the world with almost 7 million cumulative deaths worldwide. It is therefore of high importance to prepare preventative measures to aid the handling of emerging viral threats before a new pandemic is realized. One of the more prevalent and rapidly spreading viruses today are the Dengue virus (DENV). When looking closer at the viral cascade involved in infecting the host, one enzyme plays an especially important role - the NS2B-NS3 protease. Previous endeavors in the field of bioinformatics proved that the addition of a histidine-tag, that is used for purification, may be problematic in the case of the NS2B-NS3, as the tag seem to exhibit inhibitory effects on the activity of the enzyme. Based on these results, the aim of this study was to produce recombinant NS2B-NS3 with a TEV protease cleavage site added between the his-tag and the protease, allowing the removal of the his-tag through digestion with TEV protease. An attempt was also made to purposefully inhibit the activity of the NS2B-NS3, as inhibition of these viral enzymes may prove useful in the development of a possible anti-viral drug. Based on that premise, production was followed by in-silico and in-vitro docking experiments of 3 different ligands, a peptide derived from the Chenopodium quinoa plant, carvacrol and chlorogenic acid. The synthetic quinoa-peptide was unfortunately not soluble and where therefore only studied in-silico. The results showed that the production of recombinant NS2B-NS3 can be optimized to reach a higher degree of activity by removing the his-tag by TEV protease digestion. The NS2B-NS3 with his-tag exhibited a Vmax of 1.512 ± 0.103 µM/s, a Km of 0.735 ± 0.015 mM and a kcat of 0.304 ± 0.021 µM/s while the NS2B-NS3 without his-tag exhibited a Vmax of 2.127 ± 0.104µM/s, a Km of 0.940 ± 0.025 mM and a kcat of 0.375 ± 0.018 s-1, using DL-BAPNA as substrate. Carvacrol did not seem to exhibit any inhibitory effects on the NS2B-NS3, whereas chlorogenic acid seemed to be a potential inhibitor, decreasing the enzyme activity by ~24% at a concentration of 10 µM, resulting in a Vmax,app of 1.628 µM/s, a Km,app of 0.672 mM. and a kcat,app of 0.286 s-1. (Less)
Please use this url to cite or link to this publication:
author
Hallberg, Isak LU
supervisor
organization
course
KBTM05 20241
year
type
H2 - Master's Degree (Two Years)
subject
keywords
DENV NS2B-NS3, TEV protease, viral protease inhibitor, bioinformatics, recombinant protein production, docking simulations, DL-BAPNA, enzyme inhibition, AutoDock VINA, ChimeraX, AlphaFold, Quinoa, Carvacrol, Chlorogenic acid, biotechnology
language
English
id
9165376
date added to LUP
2024-06-20 14:57:29
date last changed
2024-06-20 14:57:29
@misc{9165376,
  abstract     = {{The recent 2019 COVID pandemic elucidated the tremendous effects a viral outbreak can have on the societies of the world with almost 7 million cumulative deaths worldwide. It is therefore of high importance to prepare preventative measures to aid the handling of emerging viral threats before a new pandemic is realized. One of the more prevalent and rapidly spreading viruses today are the Dengue virus (DENV). When looking closer at the viral cascade involved in infecting the host, one enzyme plays an especially important role - the NS2B-NS3 protease. Previous endeavors in the field of bioinformatics proved that the addition of a histidine-tag, that is used for purification, may be problematic in the case of the NS2B-NS3, as the tag seem to exhibit inhibitory effects on the activity of the enzyme. Based on these results, the aim of this study was to produce recombinant NS2B-NS3 with a TEV protease cleavage site added between the his-tag and the protease, allowing the removal of the his-tag through digestion with TEV protease. An attempt was also made to purposefully inhibit the activity of the NS2B-NS3, as inhibition of these viral enzymes may prove useful in the development of a possible anti-viral drug. Based on that premise, production was followed by in-silico and in-vitro docking experiments of 3 different ligands, a peptide derived from the Chenopodium quinoa plant, carvacrol and chlorogenic acid. The synthetic quinoa-peptide was unfortunately not soluble and where therefore only studied in-silico. The results showed that the production of recombinant NS2B-NS3 can be optimized to reach a higher degree of activity by removing the his-tag by TEV protease digestion. The NS2B-NS3 with his-tag exhibited a Vmax of 1.512 ± 0.103 µM/s, a Km of 0.735 ± 0.015 mM and a kcat of 0.304 ± 0.021 µM/s while the NS2B-NS3 without his-tag exhibited a Vmax of 2.127 ± 0.104µM/s, a Km of 0.940 ± 0.025 mM and a kcat of 0.375 ± 0.018 s-1, using DL-BAPNA as substrate. Carvacrol did not seem to exhibit any inhibitory effects on the NS2B-NS3, whereas chlorogenic acid seemed to be a potential inhibitor, decreasing the enzyme activity by ~24% at a concentration of 10 µM, resulting in a Vmax,app of 1.628 µM/s, a Km,app of 0.672 mM. and a kcat,app of 0.286 s-1.}},
  author       = {{Hallberg, Isak}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Production of recombinant NS2B-NS3 in E. coli and inhibition studies}},
  year         = {{2024}},
}