Directly to content
  1. Publishing |
  2. Search |
  3. Browse |
  4. Recent items rss |
  5. Open Access |
  6. Jur. Issues |
  7. DeutschClear Cookie - decide language by browser settings

The P. vivax merozoite surface protein 1 (MSP-1): Development of a recombinant protein production process and structural characterisation of MSP-1

Marzluf, Tanja

[img] PDF, English
Download (38MB) | Terms of use

Citation of documents: Please do not cite the URL that is displayed in your browser location input, instead use the DOI, URN or the persistent URL below, as we can guarantee their long-time accessibility.


Malaria in humans is most often caused by either Plasmodium falciparum or P. vivax. Research efforts have mostly focused on P. falciparum, despite P. vivax being the most widespread Plasmodium species. This parasite species differs from P. falciparum in some key aspects like its ability to form dormant liver forms, an early appearance of gametocytes in the blood stream and its strict tropism to reticulocytes. Although P. vivax and P. falciparum invade different developmental stages of red blood cells, both express a highly abundant protein on the surface of their merozoites, the merozoite surface protein 1 (MSP-1). Initially expressed as a precursor protein, MSP-1 undergoes proteolytic processing and is cleaved into four processing fragments called p83, p30, p38 and p42. These fragments remain non-covalently associated and are attached to the surface of the merozoite via a GPI anchor at the C-terminal fragment p42. MSP-1 is considered a promising vaccine candidate and P. falciparum MSP-1D is currently tested as a vaccine in a clinical trial. However, no similar vaccine was developed for P. vivax yet. The establishment of a protein production process for P. vivax MSP-1 (PvMSP-1) was an essential part of this study. Two different methods were developed, which both use individually expressed fusion proteins to assemble the full length MSP-1 protein. One fusion protein consists of the fragments p83 and p30, the other includes p38 and p42. In the first approach, the two halves are refolded together and purified by ion exchange chromatography and size exclusion chromatography. This protocol was also applied to produce P. berghei MSP-1 and test whether the assembly of a hybrid protein consisting of one P. falciparum half and one P. vivax half is possible. In the second approach contaminants are first removed from the individual fusion proteins by high resolution ion exchange chromatography before reconstitution of MSP-1. Both techniques result in a pure MSP-1 preparation, but the second approach results in a higher yield. The produced recombinant proteins were structurally characterised using a combination of bioinformatical, biophysical and biochemical methods. All recombinant proteins were cleaved by P. falciparum SUB1, indicating a high conservatism of the cleavage motifs. CD spectroscopy showed a mostly alpha-helical structure and only partial unfolding of the protein at high temperatures. Interestingly, this unfolding increased when MSP-1D was processed by SUB1 before performing thermal denaturation. This suggests an increased stability of the MSP-1 heterodimer compared to the complex consisting of four processing fragments. Prediction of the secondary structure revealed unstructured regions in all analysed MSP-1 proteins. The function of these regions remains unknown, but these sites could be responsible for interactions with other proteins. Cross-linking of MSP-1 paired with mass spectrometry uncovered a previously unknown interaction between the p83 and p42 processing fragment, leading to an updated model of MSP-1 with a globular instead of asymmetrical shape. This project provides two production processes for recombinant P. vivax MSP-1, which is now available for further immunological studies. The structural information acquired with this and other MSP-1 proteins can be used as a base to further elucidate its function and three-dimensional structure.

Item Type: Dissertation
Supervisor: Lanzer, Prof. Dr. Michael
Date of thesis defense: 15 November 2018
Date Deposited: 23 Nov 2018 09:04
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
About | FAQ | Contact | Imprint |
OA-LogoDINI certificate 2013Logo der Open-Archives-Initiative