Browsing by Author "Woodgate, Ryan."

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Recombinant expression, purification and characterisation of TzCATB and TzstefinB from Trichinella zimbabwensis.
(2024) Woodgate, Ryan.; Coetzer, Theresa Helen Taillefer.
Parasitic roundworms of the genus Trichinella, the causative agents of Trichinellosis, are highly successful parasites evident by their broad geographical and host ranges and their ability to manipulate the host immune system. The focal species of this study was T. zimbabwensis; a lesser studied member of the genus prevailing in Sub-Saharan Africa. Although not yet reported in humans, in the absence of a diagnostic test for this species of Trichinella, it poses a risk to human health and economic well-being. Research on different antigens expressed across all the stages of the parasite lifecycle may identify diagnostic, drug and vaccination targets. Cysteine proteases and protease inhibitors are particularly valuable targets for these purposes due to their essential functions in the parasite’s lifecycle, such as penetration of the small intestinal wall, nutrient acquisition and immune evasion amongst others. The aim of the present study was to recombinantly express and characterise cathepsin B (TzCATB) and stefin B (TzstefinB) from T. zimbabwensis. Recombinant TzCATB was expressed in the pET-28a and pCold-1 expression vectors as insoluble proteins at 43 and 42 kDa, respectively. After solubilisation and nickel immobilised metal affinity chromatography (Ni-IMAC) purification of these proteins, no proteolytic activity was detected. Recombinant TzCATB was subsequently expressed in the pCold-TF expression vector as a soluble protein 90 kDa protein, which was purified using immunoaffinity chromatography and the attached trigger factor fusion protein removed with thrombin cleavage. However, this preparation of TzCATB also did not have hydrolytic activity but was detected by chicken anti-TzCATB IgY antibodies in a western blot, but not by rabbit anti-human cathepsin B antibodies. Recombinant TzstefinB was expressed in the pET-28a expression vector as a soluble 16 kDa protein and purified with IMAC. Purified TzstefinB was used to raise IgY antibodies in chickens, which were confirmed to detect the protein in ELISA and western blotting. These antibodies did not detect sheep stefin B. Purified TzstefinB inhibited the hydrolysis of Z-Phe-Arg-AMC by the plant cysteine protease papain as well as by cathepsin L from the parasite Trypanosoma congolense. The inhibition of papain by TzstefinB was also detected with reverse zymography. The inhibitory activity of recombinant TzstefinB was shown to be stable from pH 5.0 to pH 9.0, was rapidly inactivated with incubation at 99 ºC, and was retained after storage at both 4 ºC and room temperature for up to 3 weeks. Thus, TzCATB was recombinantly expressed but was inactive, TzstefinB was recombinantly expressed and demonstrated to have inhibitory activity that is stable across a broad pH range but sensitive to high temperature, and both of these proteins have potential as diagnostic markers.