Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.
BACKGROUND
The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.


OBJECTIVE
To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).


METHODS
The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.


RESULTS
The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.


CONCLUSIONS
The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.
Laboratorio de Investigaciones Básicas en Bioquímica, Departamento de Química, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, D.C., Colombia Grupo de Bioquímica y Biología Celular, Instituto Nacional de Salud, Bogotá, D.C., Colombia Laboratorio de Parasitología Molecular, Universidad El Bosque, Bogotá, D.C., Colombia. aguerra@ins.gov.co.
Ángela Patricia
ÁP
Guerra
Eliana Patricia
EP
Calvo
Moisés
M
Wasserman
Jacqueline
J
Chaparro-Olaya
D000818:Animals; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D010963:Plasmodium falciparum / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
Escherichia coli.; Plasmodium falciparum; recombinant proteins