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Expression and one-step purification of Plasmodium proteins in dictyostelium
M X van Bemmelen1, C Beghdadi-Rais, C Desponds
1Institut de Biologie Cellulaire et de Morphologie, Université de Lausanne, Switzerland.
Abstract:
Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.
Insights
Slime mold expression in Dictyostelium discoideum yields high levels of Plasmodium falciparum, P. yoelii, and P. vivax malaria proteins. A simple affinity purification method facilitates large-scale production for malaria vaccine research.
Area of Science:
- Biotechnology
- Molecular Biology
- Parasitology
Background:
- Malaria remains a significant global health challenge, necessitating the development of effective vaccines.
- Recombinant protein expression is crucial for vaccine development, but challenges in yield and purification often hinder progress.
Purpose of the Study:
- To develop a robust and scalable method for expressing and purifying Plasmodium parasite proteins, specifically Circumsporozoite protein (CSP) and MSP1, for potential vaccine applications.
- To evaluate the utility of the slime mold Dictyostelium discoideum as a host for recombinant protein production.
Main Methods:
- Expression of full-length and truncated Plasmodium (P. falciparum, P. yoelii, P. vivax) proteins using Discoidin-tag expression vectors in Dictyostelium discoideum.
- Purification of fusion proteins via affinity chromatography utilizing Discoidin Ia's galactose binding activity on Sepharose-4B.
- Cleavage of the Discoidin-tag using a thrombin recognition site and analysis of protein secretion pathways (ER-independent vs. ER-dependent).
Main Results:
- High yields of recombinant Plasmodium proteins were achieved in Dictyostelium discoideum, ranging from 0.08 to 3 mg/L.
- A nearly single-step purification procedure based on affinity chromatography was successfully established.
- ER-independent secretion resulted in non-glycosylated proteins, while ER-routing led to glycosylation and retention; native conformation of P. vivax MSP1 was confirmed by patient sera.
- The purification method demonstrated efficiency and scalability for various Plasmodium polypeptides.
Conclusions:
- Dictyostelium discoideum is a viable and efficient host for high-yield expression of Plasmodium parasite proteins.
- The developed Discoidin-tag based affinity purification strategy offers a simplified and scalable approach for producing malaria vaccine candidates.
- This method has the potential to significantly facilitate the large-scale purification of Plasmodium polypeptides for research and vaccine development.