Small-scale Optimization of Recombinant Plasmodium knowlesi Apical Membrane Antigen 1 (rPkAMA1) Expression in Pichia pastoris

• Main Authors: Hoong, Chew Ching, Fatimah Nadiah, Haron, Aina, Azazi, Chua, Kek Heng, Lim, Yvonne Ai Lian, Lee, Ping Chin
• Format: Article
• Language: English
• Published: Penerbit Universiti Sultan Zainal Abidin 2020
• Subjects: QH301 Biology
• Online Access: http://eprints.unisza.edu.my/7491/1/FH02-FSK-20-48832.pdf; http://eprints.unisza.edu.my/7491/
• Institution: Universiti Sultan Zainal Abidin

Plasmodium knowlesi is the fifth human malaria parasite widespread in the forested area of Southeast Asia. In Malaysia, there were increasing cases of this zoonotic knowlesi malaria from 1,600 to over 4,000 incidences from 2016 to 2018, with 12 deaths occurred in 2018. Plasmodium species are obligate intracellular parasites and apical membrane antigen 1 (AMA1) is one of the most prominent merozoite surface protein and pivotal antimalarial drug targets, which plays a crucial role in parasites-host cells invasion. The production of the soluble and functional active recombinant protein is essential for downstream immunogenic and functional assays. Hence, the present study aims to optimize the conditions for heterologous expression of an ectodomain of P. knowlesi AMA1 (PkAMA1) protein using the Pichia pastoris system. Codon-optimized plasmid designated as PkAMA1-pPICZαA was constructed and then cloned into Escherichia coli TOP10F’ as the amplification host followed by P. pastoris KM71H as the expression host. Generally, two expression parameters were optimized: (i) induction time based on four optical density (OD600 of 3, 4, 5 and 6) and (ii) methanol concentration for induction (0.5%, 1%, 2% and 3%). Methanol induction was maintained every 24 hours up to 144 hours. In each time point, one ml of the culture was harvested, cell pellet and supernatant fraction were then visualized using 12% SDS-PAGE followed by Western blot validation. The recombinant PkAMA1 (rPkAMA1) expression conditions were successfully optimized. A soluble form of rPkAMA1 protein with approximately 55 kDa was secreted into the buffered methanol-complex (BMMY) expression medium. KM71H strain expressed a high level of rPkAMA1 protein and low endogenous secretory proteins at the OD600 4 of induction time with 2% methanol concentration for induction. Large-scale expression of rPkAMA1 will be carried out and the purified protein will be used for the downstream immunogenic assay.