Production of recombinant DNA fragmentation factor 40 in fusion to an antimicrobial peptide from spider venom and evaluation of its cytotoxic effects

Abstract

Background and purpose: DNA fragmentation factor 40 (DFF40) as an apoptotic molecule can represent a novel approach to cancer treatment. Lycosin-I (LYC-I), a peptide derived from spider venom, was considered for the targeted delivery of DFF40 to cancer cells. This study attempted to produce soluble DFF40-LYC-I and evaluate its selective lethal effects on HeLa cells.

Experimental approach: pTWINl vector was used to produce LYC-I and DFF40-LYC-I in E. coli BL21 (DE3) fused to inteins 1 and 2. IPTG concentration and incubation temperature were optimized to achieve the highest level of soluble product. To remove inteins 1 and 2 from the recombinant peptide or protein, pH shift and dithiothreitol were used for a 24-h incubation period at room temperature, respectively. MTT assay was performed to assess the biological effects of these bio-molecules on HeLa and HUVEC cell lines.

Findings/results: LYC-I and DFF40-LYC-I were detected in SDS-PAGE with bands of approximately 57 and 97 kDa, respectively. Furthermore, the 3 and 43 kDa bands showed the purified molecules. The IC₅₀ value of DFF40-LYC-I and DFF40 was determined as 6.6 and 17.03 μg/mL for HeLa, respectively. LYC-I had no cytotoxic effects on both cell lines, even at high concentrations.

Conclusion and implications: A new fusion protein with targeted cancer treatment potential was produced for the first time by LYC-I with a safe profile on normal cells. This fusion protein exhibited higher cytotoxic effects in cancer cells compared to normal cells. However, additional investigations are required to determine the apoptosis induction and evaluate selective toxicity against other cancer and normal cell lines.

Keywords: DFF40; DFF40-LYC-I; Lycosin-I; Targeted therapy..

Fig. 1.

In silico design of DFF40-LYC-I fusion protein. (A) Predicted three-dimensional structure of fusion protein after the visualization by PyMol; (B) local energy distribution of the predicted protein data bank from protein structure analysis web server; (C) Rhamachandran plot analysis of predicted protein. DFF, DNA fragmentation factor; LYC-I, lycosin-I.

Fig. 2.

Different conditions of total expression of recombinant proteins. (A) Recombinant LYC-I peptide in fusion to inteins 1 and 2. Lane 1, unstained protein ladder 26614 PageRuler™(Thermoscietific, USA); lane 2, total E. coli proteins induced by 0.1 mM IPTG at 7 °C; lane 3, total E. coli proteins induced by 0.3 mM IPTG at 7 °C; lane 4, total E. coli proteins induced by 0.5 mM IPTG at 7 °C; lane 5, total E. coli proteins induced by 0.1 mM IPTG at 22 °C; lane 6, total E. coli proteins induced by 0.3 mM IPTG at 22 °C; lane 7, total E. coli proteins induced by 0.5 mM IPTG at 22 °C; lane 8, total E. coli proteins induced by 0.1 mM IPTG at 37 °C; lane 9, total E. coli proteins induced by 0.3 mM IPTG at 37 °C; lane 10, total E. coli proteins induced by 0.5 mM IPTG at 37 °C; (B) recombinant DFF40-LYC-I in fusion to inteins 1 and 2. Lanes 1 and 8, unstained protein ladder 26614 PageRuler™; lane 2, total E. coli proteins induced by 0.1 mM IPTG at 22 °C; lane 3, total E. coli proteins induced by 0.3 mM IPTG at 22 °C; lane 4, total E. coli proteins induced by 0.5 mM IPTG at 22 °C; lane 5, total E. coli proteins induced by 0.1 mM IPTG at 7 °C; lane 6, total E. coli proteins induced by 0.3 mM IPTG at 7 °C; lane 7, total E. coli proteins induced by 0.5 mM IPTG at 7 °C; lane 9, total E. coli proteins induced by 0.1 mM IPTG at 37 °C; lane 10, total E. coli proteins induced by 0.3 mM IPTG at 37 °C; lane 11, total E. coli proteins induced by 0.5 mM IPTG at 37 °C. LYC-I, lycosin-I; E. coli, Escherichia coli; IPTG, isopropyl β-d-1-thiogalactopyranoside; DFF, DNA fragmentation factor.

Fig. 3.

Different conditions of the soluble expression of recombinant proteins. (A). LYC-I peptide in fusion to inteins 1 and 2. Lane 1, unstained protein ladder 26614 PageRuler™; Lane 2, expression of E. coli protein solution induced by 0.1 mM IPTG at 7 °C; Lane 3, expression of E. coli protein solution induced by 0.3 mM IPTG at 7 °C; Lane 4, expression of E. coli protein solution induced by 0.5 mM IPTG at 7 °C; lane 5, expression of E. coli protein solution induced by 0.1 mM IPTG at 22 °C; lane 6, expression of E. coli protein solution induced by 0.3 mM IPTG at 22 °C; lane 7, expression of E. coli protein solution induced by 0.5 mM IPTG at 22 °C; lane 8, expression of E. coli protein solution induced by 0.1mM IPTG at 37 °C; lane 9, expression of E. coli protein solution induced by 0.3 mM IPTG at 37 °C; lane 10, expression of E. coli protein solution induced by 0.5 mM IPTG at 37 °C; (B) DFF40-LYC-I in fusion to inteins 1 and 2. Lanes 1 and 8, unstained protein ladder 26614 PageRuler™; lane 2, expression of E. coli proteins induced by 0.1 mM IPTG at 7 °C; lane 3, expression of E. coli proteins induced by 0.3 mM IPTG at 7 °C; lane 4, expression of E. coli proteins induced by 0.5 mM IPTG at 7 °C; lane 5, expression of E. coli proteins induced by 0.1 mM IPTG at 15 °C; lane 6, expression of E. coli proteins induced by 0.3 mM IPTG at 15 °C; lane 7, expression of E. coli proteins induced by 0.5 mM IPTG at 15 °C; lane 9 expression of E. coli proteins induced by 0.1 mM IPTG at 37 °C; lane 10, expression of E. coli proteins induced by 0.3 mM IPTG at 37 °C; lane 11, expression of E. coli proteins induced by 0.5 mM IPTG at 37 °C. LYC-I, lycosin-I; E. coli, Escherichia coli; IPTG, isopropyl β-d-1-thiogalactopyranoside; DFF, DNA fragmentation factor.

Fig. 4.

Purification of peptide and protein using the IMPACT system. (A). Purified LYC-I. Lane 1, low range unstained protein ladder 26635 (Thermoscientif, USA); lane 2, elution of LYC-I removed from the column; (B) purified DFF40-LYC-I. Lane 1, unstained protein ladder 26612 (Thermoscientif, USA); lane 2, elution of DFF40-LYC-I removed from the column. IMPACT, intein mediated purification with an affinity chitin-binding tag; LYC-I, lycosin-I; DFF, DNA fragmentation factor.

Fig. 5.

Cytotoxicity assays of DFF40, LYC-I and DFF40-LYC-I against different cell lines after 48 h of incubation. (A) HeLa cell line; (B) HUVEC cell line. Data were presented as mean ± SD (n=3). ^*P . 0.05, ^**P . 0.01, and ^***P . 0.001 represent significant differences than DFF40 in the same concentrations. ^#P . 0.05 and ^###P . 0.001 represent significant differences versus negative control. LYC-I, lycosin-I; DFF, DNA fragmentation factor.