Uttryck av cysteineproteaser HRV 3C, sortase A och TEV på ytan av prokaryota värdceller
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Display of cysteine proteases HRV 3C, sortase A and TEV on prokaryotic hosts (English)
Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence of activity.
Place, publisher, year, edition, pages
Cysteine proteases, E.coli display, FACS, HRV 3C, sortase A, Staphylococcal display, TEV
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-172784OAI: oai:DiVA.org:kth-172784DiVA: diva2:849460