# Expression and purification of E140 protein antigen fragments of Plasmodium vivax and Plasmodium berghei for serological assays

## TL;DR

This paper describes a protocol for producing and purifying E140 protein fragments from two Plasmodium species to test immune responses in potential malaria vaccines.

## Contribution

The study introduces a reproducible method for expressing and purifying E140 protein fragments from Plasmodium berghei and Plasmodium vivax for use in serological assays.

## Key findings

- E140 protein fragments from Plasmodium berghei and Plasmodium vivax were successfully expressed and purified.
- The purified proteins were used in ELISA to detect antigen-specific antibody responses in vaccinated mice.
- The protocol includes steps for expression, solubilization, refolding, purification, and quality control of the E140 fragments.

## Abstract

Malaria, a life‐threatening disease caused by Plasmodium parasites, continues to pose a significant global health threat, with nearly 250 million infections and over 600 000 deaths reported annually by the WHO. Fighting malaria is particularly challenging partly due to the complex life cycle of the parasite. However, technological breakthroughs such as the development of the nucleoside‐modified mRNA lipid nanoparticle (mRNA‐LNP) vaccine platform, along with the discovery of novel conserved Plasmodium antigens such as the E140 protein, present new opportunities in malaria prevention. Importantly, production of recombinant proteins for malaria vaccine evaluation by serological assays often represents an additional hurdle because many Plasmodium proteins are complex and often contain transmembrane domains that make production and purification particularly difficult. This research protocol provides a step‐by‐step guide for the production and purification of P. berghei and P. vivax E140 protein fragments that can be used to test humoral immune responses against this novel malaria vaccine target. We demonstrate that the purified proteins can be successfully used in enzyme‐linked immunosorbent assay (ELISA) to evaluate antigen‐specific binding antibody responses in sera obtained from E140 mRNA‐LNP‐vaccinated mice. Therefore, these proteins can contribute to the development and evaluation of E140‐based malaria vaccines.

We provide a step‐by‐step guide for producing E140 antigen fragments from Plasmodium berghei (Pb1) and Plasmodium vivax (Pv1). Pb1/Pv1 are expressed in E. coli, solubilized by freeze–thawing, refolded by slow dilution, purified by affinity chromatography (IMAC), then concentrated and subjected to quality control. The intact proteins can be used in immunological assays (ELISA) to test antibody‐containing samples from vaccinated mice.