Optimized expression of <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 domains in <it>Escherichia coli</it>

• Main Authors: Chene Arnaud, Ahuja Sanjay, Flick Kirsten, Bejarano Maria, Chen Qijun
• Format: Article
• Language: English
• Published: BMC 2004-12-01
• Series: Malaria Journal
• Online Access: http://www.malariajournal.com/content/3/1/50

<p>Abstract</p> <p>Background</p> <p>The expression of recombinant proteins in <it>Escherichia coli </it>is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the <it>Plasmodium falciparum </it>genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of <it>P. falciparum </it>derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells.</p> <p>Methods</p> <p>Several domains of PfEMP1 genes obtained from different <it>P. falciparum </it>strains were expressed in <it>E. coli </it>as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage.</p> <p>Results and conclusions</p> <p>When expressed in <it>E. coli </it>recombinant proteins derived from <it>P. falciparum </it>sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for <it>E. coli </it>had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.</p>