Open Access
Title: Optimization of Andes virus cap-snatching endonuclease heterologous expression and purification
Language: English
Authors: Di Fabrizio, Bianca 
Issue Date: 2021
The Andes virus (ANDV) is endemic in South America and causes hantavirus cardiopulmonary syndrome (HCPS) with a case fatality rate of up to 40 %. The N-terminal Cap-ENDO domain of the L-protein of ANDV avails a cap-snatching mechanism that is necessary for viral survival. As such, this domain can be used as a target to elucidate potential medical treatments.
The wild type ANDV Cap-ENDO shows toxic activity in expression hosts, making it difficult to perform small molecules screening campaigns. This work aims to produce a target that could be exploited for drug discovery. The attenuated mutant (ANDVL1-200 N167A) was used for expression in Lemo21(DE3) Eschericia coli (E. coli) cells. To purify the ANDVL1-200 N167A protein, various methods, such as affinity chromatography, tag-removal via digestion reaction and ion exchange chromatography were performed. Assessment of the purification process was carried out by 12 % SDS-PAGE. Improvement in ANDVL1-200 N167A protein expression levels was achieved by expressing the protein with a N-terminal His-GST-3C tag. Off-column cleavage showed best results to cleave the fusion tag from the protein. To prevent the ANDVL1-200 N167A protein from precipitation during cleavage at pH ranging its isoelectric point, the 3C cleavage site was successfully exchanged by a thrombin cleavage site via a two-step PCR mutagenesis followed by In-Fusion cloning. It is proposed that thrombin can work at acidic pH and this could increase the yield recovery of ANDVL1-200 N167A after cleavage, thus improving the target´s production. In summary, this work has evaluated different steps in the purification process of a promising viral target and generated a new construct that may increase ANDVL1-200 N167A yields.
Institute: Fakultät Life Sciences 
Type: Thesis
Thesis type: Bachelor Thesis
Advisor: Cornelissen, Gesine 
Referee: Fernández-García, Yaiza 
Appears in Collections:Theses

Files in This Item:
File Description SizeFormat
DiFabrizioBiancaBA_geschwärzt.pdf10.77 MBAdobe PDFView/Open
Show full item record

Page view(s)

checked on Dec 3, 2021


checked on Dec 3, 2021

Google ScholarTM


Note about this record


Items in REPOSIT are protected by copyright, with all rights reserved, unless otherwise indicated.