Characterization of the functional domains of FKBP35 from Plasmodium Knowlesi

• Main Author: Goh, Carlmond Kah Wun
• Format: Thesis
• Language: English
• Published: 2019
• Subjects: QD Chemistry
• Online Access: https://eprints.ums.edu.my/id/eprint/25219/1/Characterization%20of%20the%20functional%20domains%20of%20FKBP35%20from%20Plasmodium%20Knowlesi.pdf; https://eprints.ums.edu.my/id/eprint/25219/

High incident of Plasmodium knowlesi accompanied by the increase in drug resistance cases in Malaysia Borneo urges us to develop a novel antimalarial drug with no resistance. Despite its advantages as an antimalarial compound without resistance effects, FK506 displayed an immunosuppressive side effect. In attempts to find FK506 replacers, solid fundamental studies on target molecule of FK506 are needed to provide a platform for the development of novel antimalarial drug without the risk of resistance. Previous studies revealed FK506 inhibited function of FKBP35 from P/asmodium fa/ciparum. Genome sequence of P. knowlesi suggested the presence of its FKBP35 (Pk-FKBP35) in which has high sequence similarity with FKBP35 from P. fa/ciparum (PF-FKBP35) and P. vivax (Pv-FKBP35), thus suggesting Pk-FKBP35 is considered a viable target for combating P. knowlesi. FKBP35 is a member of peptidyl-prolyl as-trans isomerase (PPiase), consisting FK506-binding domain (FKBD) followed by tetratricopeptide repeated domain (TPRD). There is no study so far on Pk-FKBP35, particularly on the functionality of these domains. In fact, most of the studies on Pf- and Pv-FKBP35 were focused on FKBD region. This is due to the finding that FKBD is the region where FK506 binds and inhibits PPiase catalytic function of this protein. Previous studies on PF-FKBP35 and Pv-FKBP35 also suggested that this protein might exhibit a dual-function of foldase and chaperone­like activities. However, involvement of the domains of FKBP35 on this dual­function remains poorly understood. High similarity between Pk-FKBP35 and PF­FKBP35 suggested that Pk-FKBP35 might also exhibit dual-function, yet no experimental evidence was reported. This study aims to understand the regulatory domains for the function of FKBP35 from Plasmodium knowlesi. To address, expression system containing gene encoding full-length of Pk-FKBP35 and its derivatives, Pk-FKBD and PK-TPRD were constructed. Each of them was expressed in Escherichia coli BL21(DE3). Two steps purifications including Ni-NTA binding affinity followed by gel filtration yielded 109, 162 and 189 mg of proteins from 1 L culture of Pk-FKBP35, Pk-FKBD, and Pk-TPRD respectively. Pk-FKBD showed comparable catalytic PPiase activities with full-length Pk-FKBP35 when tested using synthetic tetrapeptide (Suc-Ala-Leu-Pro-Phe-AMC) with kcav'KM of 4.7 x 105 M.1 s·1