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Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics.

Rothan HA, Ambikabothy J, Abdulrahman AY, Bahrani H, Golpich M, Amini E, A Rahman N, Teoh TC, Mohamed Z, Yusof R - PLoS ONE (2015)

Bottom Line: The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components.The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane.The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.

No MeSH data available.


Related in: MedlinePlus

Cell membrane integrity after the treatment with the peptides-fusion protein.A) TACH-MAP30-LATA induced significant LDH leakage from both cancer (approximately 80–100%) and normal cell lines (approximately 40%) at 3 μM. B) MAP30-treated cancer cells showed less than 75% of LDH leakage at 6 μM, while the normal cells showed less than 35% C) Treatment of HepG2 cells with the peptide-fusion protein, the cells showed apparent plasma membrane damage and the cellular shape was irregular with disappeared borders (arrows) compared with untreated cells (D) Untreated HepG2 cells (Two Way ANOVA, P<0.001).
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pone.0139248.g006: Cell membrane integrity after the treatment with the peptides-fusion protein.A) TACH-MAP30-LATA induced significant LDH leakage from both cancer (approximately 80–100%) and normal cell lines (approximately 40%) at 3 μM. B) MAP30-treated cancer cells showed less than 75% of LDH leakage at 6 μM, while the normal cells showed less than 35% C) Treatment of HepG2 cells with the peptide-fusion protein, the cells showed apparent plasma membrane damage and the cellular shape was irregular with disappeared borders (arrows) compared with untreated cells (D) Untreated HepG2 cells (Two Way ANOVA, P<0.001).

Mentions: To assess the cell membrane integrity after the treatment with the peptides-fusion protein, the LDH leakage was measured in cancer cell lines (HepG2 and MCF-7) and normal cell lines (WRL68 and ARPE19). Peptide-fusion protein treatment (0–6 μM) was associated with LDH leakage from cancer cells; the leakage gradually increased with TACH-MAP30-LATA concentrations, reaching approximately 80–100% at 6 μM. However, normal cells showed less than 45% of LDH leakage at similar concentrations. TACH-MAP30-LATA induced significant (Two Way ANOVA, P<0.001) LDH leakage from both cancer (approximately 80–100%) and normal cell lines (approximately 40%) at 3 μM (Fig 6A). MAP30-treated cancer cells showed less than 75% of LDH leakage at 6 μM, while the normal cells showed less than 35% (Fig 6B). In addition, no haemolysis was induced after the erythrocytes were treated with 6 μM of MAP30-LATA or MAP30 (data not shown).


Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics.

Rothan HA, Ambikabothy J, Abdulrahman AY, Bahrani H, Golpich M, Amini E, A Rahman N, Teoh TC, Mohamed Z, Yusof R - PLoS ONE (2015)

Cell membrane integrity after the treatment with the peptides-fusion protein.A) TACH-MAP30-LATA induced significant LDH leakage from both cancer (approximately 80–100%) and normal cell lines (approximately 40%) at 3 μM. B) MAP30-treated cancer cells showed less than 75% of LDH leakage at 6 μM, while the normal cells showed less than 35% C) Treatment of HepG2 cells with the peptide-fusion protein, the cells showed apparent plasma membrane damage and the cellular shape was irregular with disappeared borders (arrows) compared with untreated cells (D) Untreated HepG2 cells (Two Way ANOVA, P<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587966&req=5

pone.0139248.g006: Cell membrane integrity after the treatment with the peptides-fusion protein.A) TACH-MAP30-LATA induced significant LDH leakage from both cancer (approximately 80–100%) and normal cell lines (approximately 40%) at 3 μM. B) MAP30-treated cancer cells showed less than 75% of LDH leakage at 6 μM, while the normal cells showed less than 35% C) Treatment of HepG2 cells with the peptide-fusion protein, the cells showed apparent plasma membrane damage and the cellular shape was irregular with disappeared borders (arrows) compared with untreated cells (D) Untreated HepG2 cells (Two Way ANOVA, P<0.001).
Mentions: To assess the cell membrane integrity after the treatment with the peptides-fusion protein, the LDH leakage was measured in cancer cell lines (HepG2 and MCF-7) and normal cell lines (WRL68 and ARPE19). Peptide-fusion protein treatment (0–6 μM) was associated with LDH leakage from cancer cells; the leakage gradually increased with TACH-MAP30-LATA concentrations, reaching approximately 80–100% at 6 μM. However, normal cells showed less than 45% of LDH leakage at similar concentrations. TACH-MAP30-LATA induced significant (Two Way ANOVA, P<0.001) LDH leakage from both cancer (approximately 80–100%) and normal cell lines (approximately 40%) at 3 μM (Fig 6A). MAP30-treated cancer cells showed less than 75% of LDH leakage at 6 μM, while the normal cells showed less than 35% (Fig 6B). In addition, no haemolysis was induced after the erythrocytes were treated with 6 μM of MAP30-LATA or MAP30 (data not shown).

Bottom Line: The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components.The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane.The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.

No MeSH data available.


Related in: MedlinePlus