Production of recombinant proteins from Plasmodium falciparum in Escherichia coli

Abstract

Introduction: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). Materials and 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. Conclusion: 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.

Structural insights into Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase: oligomeric assembly

“…The identified ORF (PF3D7_1327600) was amplified by polymerase chain reaction (PCR) from 1 mg of P. falciparum (FCB2 strain) gDNA, which was extracted with proteinase K and phenol chloroform according to previously described methodologies. 21 PCR was performed using Platinum Pfx (Invitrogen, Carlsbad, USA)polymerase and the following oligonucleotides: forward 5’-CACCATGCATAAGAATATATGT-3’, reverse 5’-CTAATTAAAATCATATAAGTT-3’. The thermal cycles consisted of an initial denaturation at 94ºC for 2 min, followed by 30 cycles of denaturation at 94ºC for 15 s, annealing at 55ºC for 30 s, and extension at 68ºC for 1 min.…”

Optimization of Fermentation Conditions for Recombinant Lipase Expression At Small Scale Using Response Surface Methodology for Preliminary Production In Two Bioreactor Platforms.

“…According to Kano et al, lowering temperatures can increase recombinant protein solubility, but it is specific of each protein [57]. Similar studies have decreased the temperature from 37°C to temperatures below 20°C but without a significant increase in the solubility of different recombinant proteins produced in E. coli [32,58,59].…”

Identification of Plasmodium knowlesi Merozoite Surface Protein‐119 (PkMSP‐119) novel binding peptides from a phage display library

“…Malaria proteins are among the most difficult and challenging to express with in vitro methods due to their extreme genetic codon usage, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies. Various organisms have been tested for the production of malaria proteins, including E. coli [41,42], Pichia pastoris (yeast) [43,44], transgenic mice [45] and transgenic tobacco plants [46]. Among these, E. coli is the most frequently used expression system.…”

“…The binding signals for each peptide were estimated using absorbance value at 405 nm towards BSA (control) and rPkMSP-1 19 , respectively. [Color figure can be viewed at wileyonlinelibrary.com] insoluble inclusion bodies in bacterial cells [41,47]. The solubility of a protein correlates with its correct structure that is formed during a post-translational folding process.…”