Proteases have a fundamental role in regulating diverse biological processes and are important in the biotechnological and medical fields. Therapeutic proteases have great potential but have been limited due to the lack of high throughput protein engineering methods. In this thesis, a method was developed for high throughput screening of protease libraries based on competitive growth in selective media. A proof-of-principle method using the Tobacco Etch Virus protease (TEVp) was developed. TEVp and the reporter consisting of an aggregation-prone peptide amyloidbeta42 (Aβ42) genetically fused to the antibiotic resistance enzyme chloramphenicol acetyltransferase (CAT), were co-expressed in Escherichia coli. The CAT enzyme makes the cells resistant to Chloramphenicol (Cml). Two different reporters containing different cleavage sites situated between the Aβ42 and CAT were used for which TEVp has distinguishable proteolysis efficiencies. Cleavage of the fusion protein would give the cell a growth advantage in media with Cml since the CAT enzyme would avoid misfolding due to Aβ42. The method demonstrates that cells with different substrates can be differentiated based on their survival. A 100-fold enrichment of clones expressing the efficient substrate was also demonstrated from a background of 1:1000 of clones expressing the inefficiently cleaved substrate. Moreover, a semi-rational TEVp library was successfully cloned and co-electroporated with the reporter into E. coli for future selection.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-278571 |
| Date | January 2020 |
| Creators | Lagebro, Vilma |
| Publisher | KTH, Skolan för kemi, bioteknologi och hälsa (CBH) |
| 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 | TRITA-CBH-GRU ; 2020:126 |
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