Purification of His-Sumo Tagged RNaseR and Its Functional Analysis
Date
2022-05
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University of Delaware
Abstract
RNaseR is an exonuclease belonging to the RNase II family that degrades linear RNA in the 3’ to 5’ direction and does not have a cleaving effect on circular RNA. The objective of this thesis is to overexpress and purify His-tagged RNaseR from recombinant E. colicells that is free of other RNases and contaminants through growing cultures, employing purification techniques such as nickel column affinity chromatography, butyl column chromatography, and Gel Filtration Chromatography (GFC). Since RNaseR is very expensive and is produced by only one biotechnology company-Lucigen Inc, which unfortunately produces inconsistent results in multiple cases, it is crucial to produce pure RNaseR in-house that is functional and free of other contaminants that could cause circularRNA degradation. Although functional RNaseR was successfully purified in one of the purification runs, it had low reproducibility and could not be replicated using the same method and reaction conditions. Multiple optimizations were made that helped to get increased expression of RNaseR and helped in understanding the process of RNaseR purification and how the protein reacts in different reaction conditions.
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Keywords
RNA, Purification, Biotechnology