Recombinant production of the Giardia intestinalis cysteine protease CP10217 in Pichia pastoris
Sammanfattning: Giardia intestinalis is one of the leading causes of diarrheal diseases, affecting about 280 million people every year. By characterizing the virulence factors of G. intestinalis, new drug targets can be found to treat giardiasis. In addition to the adhesive disc and the variant surface proteins, cysteine proteases are some of the most interesting virulence factors in Giardia. In this project, one of the major secreted cysteine proteases, CP10217, was studied. The intent was to study the structure by modelling and to characterize CP10217 by expressing it in yeast cells and purifying the supernatant by using Immobilized Metal Affinity Chromatography (IMAC). The molecular modelling showed that the Giardia CP can be modelled on the structures of human and Trypanosoma brucei CPs. The process of expressing and purifying CP10217 in this manner proved difficult. The protease seemed to be very active when expressed, probably resulting in self-cleavage into its active form and later digestion of the whole protein, leading to a low protein yield from the purification. Two approaches were tested in order to increase the protein yield. First, expression at different pH ranges, and secondly, by re-cloning CP10217 with an extra 108 bp sequence at the 5’ end. While changes in pH did not seem to affect the yield, sequencing results of the new vector showed that the cloning worked. More work on this new vector is needed to further analyse, and possibly characterize CP10217.
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