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Plasmodium falciparum surface protein 25 (Pfs25) is a hard-to-express and hard-to-solubilize protein in Escherichia coli. To overcome this problem, the phase 1 flagellin of Salmonella enterica serovar Typhimurium (FliC) was used as a fusion partner for Pfs25. The fusion expression of Pfs25 with FliC greatly enhanced the expression level and solubility of Pfs25 in E. coli BL21(DE3). The Ni-purified fusion protein of FliC-Pfs25 was recognized by two anti-Pfs25 monoclonal antibodies. By comparison, it was shown that the Pfs25 within FliC-Pfs25 contained epitopes similar or identical to those on Pichia pastoris-produced Pfs25. The data obtained from this study demonstrated that the fusion with Salmonella flagellin greatly improved the expression of Pfs25 in E. coli.
Plasmodium falciparum surface protein 25 (Pfs25) is one of the leading antigens of malaria transmission blocking vaccine, which contains 2two conserved cysteine residues maintaining a structure of 4 epidermal growth factor-like domains.1 Correct conformation is critical for recombinant Pfs25 produced in heterologous protein expression systems to induce functional antibodies against malaria parasites. Escherichia coli is a popular protein expression system due to its advantages in easy handling and inexpensive cultivation. However, studies showed that Pfs25 was not only lowly expressed but also aggregated in the form of inclusion body in E. coli.2,3 The ability of insoluble Pfs25 to induce functional antibodies was greatly reduced.2
Co-expression with a fusion partner is one of the common strategies to improve the expression of some hard-to-express and hard-to-solubilize proteins in E. coli.4,5 Many fusion partners such as E. coli maltose binding protein have the ability to increase the yield, enhance the solubility and even promote the proper folding of their fused proteins. As an agonist of innate immune system, Salmonella flagellin has been demonstrated to be a potent adjuvant for various antigens and widely tried in vaccine research and development.6 In addition, Salmonella flagellin presents some distinct properties that make it a potential fusion partner: (1) the phase 1 flagellin of Salmonella enterica serovar Typhimurium (FliC) doesn't contain any cysteine residue in its molecule, and (2) FliC can be highly expressed as a soluble protein in E. coli.7
In this study, Pfs25 was expressed in E. coli as a fusion protein with FliC. The Pfs25 gene without signal peptide and GPI-like anchor sequence was firstly PCR amplified from the genomic DNA of P. falciparum 3D7, and then inserted at the downstream of the FliC gene on the pET28a(+)-fliC plasmid (made in our previous study7) (Fig. 1A). The recombinant pET28a(+)-fliC-pfs25 plasmid was transformed into the host strain of E. coli BL21(DE3). After induction with IPTG, the fusion protein of FliC-Pfs25 was highly expressed in E. coli. SDS-PAGE of the whole cell lysate of E. coli showed a heavy protein band appeared on the gel with an estimated molecular weight of about 72 kDa (Fig. 1B, Lane 2). Compared to the single expression of Pfs25 in E. coli, which presented only a faint protein band on SDS-PAGE gel,3 the fusion expression with FliC remarkably enhanced the expression level of Pfs25 in E. coli. The solubility of FliC-Pfs25 was evidenced by the fact that a large amount of the fusion protein existed in the sonicated cell supernatant of E. coli after IPTG induction (Fig. 1B, Lane 3).
With a C-terminal 6[His] tag, the FliC-Pfs25 fusion protein was purified from the sonicated cell supernatant by Ni-Sepharose affinity chromatography. Successful purification was achieved although some aggregates were formed during the process (Fig. 1C). Western blot analysis showed that the purified protein of FliC-Pfs25 was recognized not only by anti-FliC mAb (InvivoGen) but also by 2 anti-Pfs25 monoclonal antibodies, mAb 1A6 and mAb 4D10 (kindly provided as gifts by Dr. Yimin Wu8) (Fig. 1D). When an irrelevant monoclonal antibody (mAb 5.2) was used in the analysis, no any blot could be observed.
Since yeast expression system can produce Pfs25 in its native conformation,2,9,10 the Pichia pastoris-produced Pfs25 (yPfs25) (its production will be described elsewhere) was used as a reference to assess the conformation of FliC-Pfs25. Inhibition ELISA showed that the reaction between yPfs25 and its mouse immune sera (made by immunizing BALB/c mice with yPfs25 formulated with Freund adjuvant) was apparently inhibited by FliC-Pfs25 in a dose dependent manner (Fig. 1E). When the recombinant FliC (rFliC) (made in our previous study7) was used as an inhibitor, no such inhibition was appeared. In turn, the mouse immune sera induced by FliC-Pfs25 (made by immunizing BALB/c mice with FliC-Pfs25 formulated with Alum adjuvant) recognized yPfs25 well (Fig. 1F). The mouse anti-rFliC immune sera (made in our previous study7) didn't recognize yPfs25. The results suggested that the Pfs25 as a fused protein within FliC-Pfs25 had a conformation, to some extent, similar to that of yPfs25.
Taken together, the data obtained from this study demonstrated that Salmonella flagellin was an effective fusion partner for Pfs25, which greatly improved the expression of Pfs25 in E. coli. Nonetheless, considering that Pfs25 is a complex protein, the strategy of over-expression of chaperone/ foldase in E. coli cytoplasma will be tried in the future study to further promote the correct folding of FliC-Pfs25.
No potential conflicts of interest were disclosed.
This work was supported by the grants of the National Natural Science Foundation of China (F Qian, No. 81072498) and the National Basic Research Program (973 Program) of China (H Xu, No. 2007CB513105).