Expression, purification and characterization of plasmodium falciparum acidic basic repeat antigen

• Main Author: Sek, Mun Foong
• Other Authors: Gruber, Ardina
• Format: Final Year Project
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences::Biochemistry
• Online Access: http://hdl.handle.net/10356/65359
• Institution: Nanyang Technological University

The Plasmodium falciparum acidic basic repeat antigen (ABRA) is a 101 kDa highly conserved protein found at the merozoite surface during schizont rupture and in the parasitophorous vacuole within an infected erythrocyte. It is involved in the complex invasion process of erythrocytes in the malaria life cycle by binding to Band 3 protein, an anion transporter protein located on the surface of erythrocyte. In this study, soluble construct covering the N-terminal domain of ABRA from amino acids 24 to 190 (ABRA 24-190) was expressed and purified using Nickel-nitrilotriacetic acid (Ni-NTA) and size exclusion chromatography. ABRA 24-190 exhibits auto-proteolytic activity and hence, an attempt was made to prove and characterize the type of protease activity it exhibits by using different protease inhibitors during the purification process. This study confirms that ABRA shows serine protease activity, with phenylmethylsulfonyl fluoride and cOmplete, EDTA-free cocktail inhibitor tablets showing the most inhibition of protease activity. Purified ABRA 24-190 was tested for binding to exofacial Loop 3 and Loop 7 of the human erythrocyte Band 3 protein using chromatographic and mass spectroscopy techniques, since these loops can be seen to inhibit cytoadherence. Binding of protein to peptide was observed at a 2:1 protein to peptide molar ratio.