Open Access
Title: Optimization of Andes virus cap-snatching endonuclease heterologous expression and purification
Language: English
Authors: Di Fabrizio, Bianca 
Issue Date: 2021
Abstract: 
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.
URI: http://hdl.handle.net/20.500.12738/11867
Institute: Fakultät Life Sciences 
Type: Thesis
Thesis type: Bachelor Thesis
Advisor: Cornelissen, Gesine 
Referee: Fernández-García, Yaiza 
Appears in Collections:Theses

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