The bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection worldwide. When left untreated, chlamydial infections can lead to severe complications, such as infertility. Lack in current prevention and management due to its asymptomatic course of infection highlight the need for an effective vaccine against chlamydia. There is no vaccine at present to protect against chlamydia, but research is ongoing. A research group at Örebro University has developed a protein antigen candidate. This project focused on the production of the candidate, here called Protein X, for preclinical trials. This included optimising production in Escherichia coli to maximise formation of soluble protein, optimising purification, buffer exchange and removal of His-tag. It was found that formation of soluble protein was favoured in lower expression temperatures. Furthermore, purification was performed on soluble and insoluble protein fractions using immobilised metal affinity chromatography. However, issues with inefficient binding to the resin and purity could not be solved and further optimisation is needed. Buffers were tested to find a suitable buffer for preclinical experiments, but the protein precipitated in all buffers. It was however found that protein from the insoluble fraction dissolved in pure water. Lastly, removal of the His-tag was performed with a non-enzymatic method that utilises nickel ions instead of expensive proteases. Efficient removal was however not achieved and enzymatic methods may be considered instead. In conclusion, this project highlighted issues in the production of Protein X and may guide the research group towards improving this process for efficient preclinical preparations.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-451976 |
| Date | January 2021 |
| Creators | Koivula, Therese |
| Publisher | Uppsala universitet, Molekylär biomimetik |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | UPTEC X ; 21006 |
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