Preliminary Studies on the Expression and Purification of Functionally Active Recombinant Plasmepsin 9 from Plasmodium falciparum
International Journal of Biochemistry Research & Review · pp. 107–118 · Published 2 Apr 2013
10.9734/IJBCRR/2013/3023Abstract
Aim: Plasmepsins, a group of homologous aspartic proteinases are attractive drug targets against malaria. Plasmepsin 9 (PM9) expressed in the blood stage of malaria life cycle with unknown function has been strongly considered as a potential target. However, recombinant expression of active PM9 for biochemical and structure-activity analysis of the enzyme has been very challenging. This paper presents preliminary report on the expression and purification of active recombinant plasmepsin 9 from Plasmodium falciparum. Place and Duration of Study: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida between November 2011 and October, 2012. Methodology: The plasmid of truncated proenzyme (proPfPM9) from Plasmodium falciparum was expressed in Escherichia coli Rosetta 2(DE3)pLysS competent cells. The protein was purified from inclusion bodies using combination of cation exchange and gel filtration chromatography. The expression and purification fractions were subjected to SDS-PAGE. The zymogen was activated to produce mature form of active recombinant PfPM9. Catalytic activity test of PfPM9 was determined using a chromogenic substrate, Lys-Pro-Ile-Glu-Phe-Nph*Arg-Leu (RS6). Substrate hydrolysis was examined from substrate cleavage scan. Results: SDS-PAGE confirmed the expression and purification of truncated proenzyme (proPfPM9) with molecular weight of approximately 54 kD. Kinetic assays showed that the purified PfPM9 was active. The hydrolytic activity of PfPM9 on RS6 was confirmed by a shift in absorbance peak from 280 nm to 272 nm at the end of assay reactions. Inhibition studies on the mature PfPM9 showed that the activity of the enzyme was inhibited by pepstatin A. Conclusion: Protocols for successful expression and purification of active recombinant plasmepsin 9 in E. coli have been established in this preliminary report. These will aid production of sufficient yield of PfPM9 for detailed kinetic and structural characterization of the enzyme and development of future inhibitors against malaria.
References (24)
- 1 A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding [DOI]
- 2 Genome sequence of the human malaria parasite Plasmodium falciparum [DOI]
- 3 The Transcriptome of the Intraerythrocytic Developmental Cycle of Plasmodium falciparum [DOI]
- 4 A proteomic view of the Plasmodium falciparum life cycle [DOI]
- 5 Discovery of Gene Function by Expression Profiling of the Malaria Parasite Life Cycle [DOI]
- 6 Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine [DOI]
- 7 Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets [DOI]
- 8 Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte [DOI]
- 9 Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease. [DOI]
- 10 Heterologous expression of proteins from Plasmodium falciparum: Results from 1000 genes [DOI]
- 11 Novel Uncomplexed and Complexed Structures of Plasmepsin II, an Aspartic Protease from Plasmodium falciparum [DOI]
- 12 Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum [DOI]
- 13 Heterologous expression of plasmodial proteins for structural studies and functional annotation [DOI]
- 14 Role of Plasmodium falciparum Digestive Vacuole Plasmepsins in the Specificity and Antimalarial Mode of Action of Cysteine and Aspartic Protease Inhibitors [DOI]
- 15 HIV and malaria co-infection: interactions and consequences of chemotherapy [DOI]
- 16 Crystal structures of the free and inhibited forms of plasmepsin I (PMI) from Plasmodium falciparum [DOI]
- 17 Expression and Characterisation of Plasmepsin I from Plasmodium falciparum [DOI]
- 18 Activity and inhibition of plasmepsin IV, a new aspartic proteinase from the malaria parasite, Plasmodium falciparum [DOI]
- 19 Achiral, Cheap, and Potent Inhibitors of Plasmepsins I, II, and IV [DOI]
- 20 Naturally‐occurring and recombinant forms of the aspartic proteinases plasmepsins I and II from the human malaria parasite Plasmodium f alciparum [DOI]
- 21 Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries [DOI]
- 22 Computational Perspectives into Plasmepsins Structure—Function Relationship: Implications to Inhibitors Design [DOI]
- 23 Analysis of Proteinase Specificity by Studies of Peptide Substrates [DOI]
- 24 Investigation of 2-Fold Disorder of Inhibitors and Relative Potency by Crystallizations of HIV-1 Protease in Ritonavir and Saquinavir Mixtures [DOI]
Cited by 0
No indexed citations yet.
Related research
- ABO Blood Groups and Malaria Prevalence in a Referral Hospital in Nigeria — shares topic coverage
- Prevalence of Malaria among Pregnant Women and Children Under Five Years in Abi Local Government Area, Cross River State, Nigeria — shares topic coverage
- A Study on Relationship of Thrombocytopenia and Likelihood of Severe Malaria in Patients Admitted With Malaria in a Tertiary Care Hospital of Dakshina Kannada — shares topic coverage
- Platelet Alteration in Plasmodium vivax Malaria Patients in Thailand — shares topic coverage
- Patients’ Perception of Health Care Services in Malaria Endemic Area in Mali: Villages with Health Care Services Versus villages Without Health Care Services — shares topic coverage
Article metrics
Real usage data collected on this platform.
54
Page views
0
PDF downloads
31
Outbound clicks
0
Citations
Views over time
Views by country
Approximate, from request IP at view time — not citizenship or institution. Countries with fewer than 5 views are grouped as "Other".
Traffic sources
Referring site, by host.
Views and downloads exclude known bots/crawlers. Citations combines this platform's own DOI-resolved index with each external source's own reported total — see Cited by above for individually listed citing works. Last refreshed 0 seconds ago.