Thermo Fisher Scientific in Uppsala manufactures in vitro tests for allergy diagnostics by producing recombinant allergens. However, the absence of an effective method for quantification of recombinant protein post-fermentation, challenges the production since protein yield cannot be determined. This study is made upon request by Thermo Fisher Scientific in Uppsala as a response to this issue with the aim to provide a recommendation of a method to implement. Thermo Fisher Scientific uses histidine-tagged recombinant proteins in their production which allows purification through immobilized metal ion affinity chromatography (IMAC). Hence, this study mainly surveys quantification methods that utilize the histidine-tag. This project provides insight into the following methods: SDS-PAGE, Western blotting, ELISA, Simple Western and FluoroHis-PAGE. The methods have been evaluated based on three key aspects: cost, analysis time, and method performance. The recommended method for implementation is FluoroHis-PAGE, as it was found to be the most appropriate method for Thermo Fisher Scientific’s current process. FluoroHis-PAGE utilizes fluorescent Ni-NTA or antibody conjugates, which demonstrated high performance in the quantification of His-tagged recombinant proteins at a low cost and analysis time.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-529921 |
| Date | January 2024 |
| Creators | Lindblom, Justin, Nettelbladt, Maja, Nilbrink, Adam, Oskarsson, Clara, Taheri, Mustafa, Östlund, Maija |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
| Source Sets | DiVA Archive at Upsalla University |
| Language | Swedish |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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