Graduation Year

2018

Document Type

Thesis

Degree

M.S.P.H.

Degree Name

MS in Public Health (M.S.P.H.)

Degree Granting Department

Global Health

Major Professor

John H. Adams, Ph.D.

Committee Member

Thomas Unnasch, Ph.D.

Committee Member

James Leahy, Ph.D.

Keywords

Affinity Chromatography, Heterologous Expression, Malaria, Molecular Cloning, Public Heath

Abstract

Malaria is a major threat to the public health worldwide as it is affecting populations in tropical and subtropical areas globally. Among those populations are around 40% of pregnant women and children who are susceptible to this disease. Plasmodium falciparum is the most lethal agent that causes malaria in human. Currently, there is drug resistance against antimalarial drugs in parasite against treatment of malaria infections, it is essential to search for new drug targets in order to find cure and alleviate suffering of human population.

There are approximately 100 protein kinases in P. falciparum that are involved in phosphorylation of asexual blood stage. Hence, the phosphorylation plays an important part in the development of different stages of malarial parasites. Due to their significance in the parasite life cycle, one of the protein kinase of P. falciparum belongs to the ABC-1 family of proteins. PfABCK2 can be a therapeutic target due to its higher expression during the late schizont stage of blood stage form.

The bioinformatic analysis and preliminary results of PfABCK2 showed the heterologous expression of this protein. Hence, the gene of PfABCk2 was ligated into pET21a+ vector with His-tag at C-terminus and transformed into BL-21 (DE3) competent cells that were verified through Miniprep and DNA sequencing. Furthermore, this gene construct is utilized to heterologous express this protein with IPTG and afterwards purified the recombinant protein kinase using nickel affinity chromatography as shown on 10% SDS-PAGE with the expected 36 kDa protein band. Therefore, the aim of this study is to partially characterize PfABCK2 protein kinase utilizing molecular cloning, heterologous express and protein kinase activity assay.

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