EXPRESSION OF PLASMODIUM FALCIPARUM CYSTEINE-RICH PROTECTIVE ANTIGEN (PFCYRPA) 26 - 181 FRAGMENT IN ESCHERICHIA COLI

• Main Author: Galih Prameswari, Aurelia
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/82934
• Institution: Institut Teknologi Bandung

Malaria is a diseased caused by Plasmodium sp. and transmitted through a female Anopheles mosquito bite. Malaria could lead to death so proper prevent treatment is needed, vaccine is one of the treatments. Plasmodium falciparum Cysteine-rich Protective Antigen (PfCyRPA) is one of the vaccine antigen candidate. Anti-PfCyRPA antibodies are able to inhibit parasite growth both in vitro and in vivo. This research purposes are to construct the recombinant plasmid pET-16b-PfCyRPA and to express PfCyRPA fragment 26 - 181 gene in Escherichia coli. There are 4 main steps, including are (i) PfCyRPA gene amplification using PfCyRPA genome DNA (gDNA) template from Jayapura isolate utilize Polymerase Chain Reaction (PCR) technique, (ii) pGEM-T-PfCyRPA and pET-16b-PfCyRPA recombinant plasmid construction and its successful is confirmed by colony PCR, restriction analysis, and nucleotide sequence determination using the Sanger sequencing, (iii) E. coli BL21 CodonPlus (DE3) and E. coli Arctic Express (DE3) strains are transformed with pET-16b-PfCyRPA recombinant plasmid, and lastly (iv) PfCyRPA fragments 26 - 181 recombinant protein expression in E. coli BL21 CodonPlus (DE3) RIPL and E. coli Arctic Express (DE3) and identified by SDS-PAGE and Western Blot. The result of this research is ~480 bp PfCyRPA gene was successfully amplified using annealing temperature at 54.5 °C. pGEM-T-PfCyRPA and pET-16b-PfCyRPA recombinant plasmid construction as well as E. coli BL21 CodonPlus (DE3) RIPL and E. coli Arctic Express (DE3) transformation has been done successfully. Molecular weight of PfCyRPA fragmen 26 - 181 recombinant protein is estimated to be ~20 kDa. Expression of PfCyRPA fragmen 26 - 181 in E. coli BL21 CodonPlus (DE3) RIPL was induced by 0.5 mM IPTG at 37 °C for 4 hours, meanwhile expression in E. coli Arctic Express (DE3) was induced by 0.5 mM at 10 for 16 - 18 hours. SDS-PAGE and Western Blot show that PfCyRPA fragmen 26 - 181 was expressed as inclusion bodies in E. coli BL21 CodonPlus (DE3) RIPL and was expressed as inclusion bodies and as soluble protein in E. coli Arctic Express (DE3). The results also show that PfCyRPA fragment 26 - 181 recombinant protein was expressed with higher level in E. coli BL21 CodonPlus (DE3) RIPL. Therefore, conclusions of this research are pGEM-T-PfCyRPA dan pET-16b-PfCyRPA have been constructed successfully and PfCyRPA fragment 26 - 181 was successfully expressed in E. coli BL21 CodonPlus (DE3) RIPL and E. coli Arctic Express (DE3) with a higher level expression in E. coli BL21 CodonPlus (DE3) RIPL as inclusion bodies. For further research, inclusion bodies of PfCyRPA fragment 26 - 181 is refold and the recombinant protein is purify using Ni-NTA affinity chromatography.