The identification of goat peroxiredoxin-5 and the evaluation and enhancement of its stability by nanoparticle formation

Abstract

An anticancer bioactive peptide (ACBP), goat peroxiredoxin-5 (gPRDX5), was identified from goat-spleen extract after immunizing the goat with gastric cancer-cell lysate. Its amino acid sequence was determined by employing 2D nano-LC-ESI-LTQ-Orbitrap MS/MS combined with Mascot database search in the goat subset of the Uniprot database. The recombinant gPRDX5 protein was acquired by heterogeneous expression in Escherichia coli. Subsequently, the anti-cancer bioactivity of the peptide was measured by several kinds of tumor cells. The results indicated that the gPRDX5 was a good anti-cancer candidate, especially for killing B16 cells. However, the peptide was found to be unstable without modification with pharmaceutical excipients, which would be a hurdle for future medicinal application. In order to overcome this problem and find an effective way to evaluate the gPRDX5, nanoparticle formation, which has been widely used in drug delivery because of its steadiness in application, less side-effects and enhancement of drug accumulation in target issues, was used here to address the issues. In this work, the gPRDX5 was dispersed into nanoparticles before delivered to B16 cells. By the nanotechnological method, the gPRDX5 was stabilized by a fast and accurate procedure, which suggests a promising way for screening the peptide for further possible medicinal applications.

Figure 1. NanoLC-orbitrap MSD TIC of ACBP’s digests.

NanoLC-orbitrap MSD, nano-flow liquid chromatography in tandem with orbitrap mass spectrum detection; TIC, total ion chromatogram.

Figure 2. The construction of gPRDX5-pET-28a(+) plasmid and the acquisition of heterogeneous expressed gPRDX5 protein.

(a) The DNA fragment of gPRDX5 was connected to the expression vector pET-28a(+) and the plasmid was then transformed to BL21 (DE3) for protein purification, (b) The gPRDX5 was acquired by heterogeneous expression and it molecular weight was at about 18 kDa.

Figure 3. The preliminary screening of the anticancer bioactivity of gPRDX5.

The gPRDX5 was sensitive to all the six tumor cell lines with the IC50 values at about 34.15, 30.326, 44.47, 56.985, 32.545 and 28.107 for MCF7, A549, HCT116, Hep G2, Hela and B16 cells, respectively (P < 0.05). The sensitivity was the highest against B16 cells. The values varied from sample to sample within the same tumor cells.

Figure 4. Characterizations of gPRDX5 nanoparticles.

(a) The typical gPRDX5 NPs by TEM with the diameters ranging from 30 to 70 nm. (b) The average size was at about 50 nm. (c) The NPs remained stable in size within a month.

Figure 5. The anticancer effect of gPRDX5-NPs on the B16 cells.

(a) The CLSM images of the internalized NPs in B16 cells. (b) The cytotoxicity of PBS, gPRDX5 and gPRDX5-NPs (P < 0.05). The gPRDX5-NPs was more sensitive against the B16 cells.