Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli.
BACKGROUND
The expression of recombinant proteins in Escherichia coli 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 Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells.


METHODS
Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli 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.


CONCLUSIONS
When expressed in E. coli recombinant proteins derived from P. falciparum 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 E. coli 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.
Flick
Kirsten
K
Microbiology and Tumor Biology Centre (MTC), Karolinska Institutet, Stockholm, Sweden. Kirsten.Flick@smi.ki.se
Ahuja
Sanjay
S
Chene
Arnaud
A
Bejarano
Maria Teresa
MT
Chen
Qijun
Q
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression / Q000235:genetics*; D018503:Genome, Protozoan / Q000235:genetics; D006493:Heparin / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D010963:Plasmodium falciparum / Q000737:chemistry / Q000235:genetics*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D000953:Antigens, Protozoan; D003062:Codon; C066001:Duffy antigen binding protein, Plasmodium; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C085731:erythrocyte membrane protein 1, Plasmodium falciparum; D006493:Heparin