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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

Purification of inclusion bodies by solubilization and refolding in alkaline-based buffer containing redox agents.A) Semi-solubilization of inclusion bodies in dH2O (pH 12) after 10 min of incubation. L1, the isolated inclusion bodies; L2, purified protein (final product); L3, removing of host cell proteins; L4, remaining insoluble aggregates after final solubilization (each line duplicated). B) Refolding of soluble protein in buffer containing oxidized and reduced glutathione to reform the disulphide bridges of TACH. The refolded protein showed different levels under reducing and non-reducing conditions of the SDS-PAGE, indicating the formation of disulphide bridges. C) SDS-PAGE analysis of the fusion protein. L1, a single band at the expected size for the monomer. L2, double bands of the fusion protein. The upper faint band at the dimer size and the lower thick band at the monomer size (arrows).
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pone.0139248.g003: Purification of inclusion bodies by solubilization and refolding in alkaline-based buffer containing redox agents.A) Semi-solubilization of inclusion bodies in dH2O (pH 12) after 10 min of incubation. L1, the isolated inclusion bodies; L2, purified protein (final product); L3, removing of host cell proteins; L4, remaining insoluble aggregates after final solubilization (each line duplicated). B) Refolding of soluble protein in buffer containing oxidized and reduced glutathione to reform the disulphide bridges of TACH. The refolded protein showed different levels under reducing and non-reducing conditions of the SDS-PAGE, indicating the formation of disulphide bridges. C) SDS-PAGE analysis of the fusion protein. L1, a single band at the expected size for the monomer. L2, double bands of the fusion protein. The upper faint band at the dimer size and the lower thick band at the monomer size (arrows).

Mentions: To retrieve the bioactivity of the recombinant peptide-fusion protein, pure inclusion bodies were solubilized and refolded in alkaline-based buffer containing redox agents. Although the inclusion bodies were washed extensively, the host cells contaminants were retained after refolding. Therefore, the purified inclusion bodies were washed with dH2O (pH 12) after 10 min of incubation. This step is important to solubilize the host cell proteins that may aggregate with the inclusion bodies; the collected supernatant showed only the host cell contaminants (Fig 3A). The final solubilization of the inclusion bodies was achieved at pH 12.5 in the presence of 2 M urea. The remaining inclusion bodies were insoluble even with higher concentrations of urea, showing that the recombinant peptide-fusion protein was approximately 60% of the total inclusion bodies as shown in Fig 2C and Fig 3A.


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)

Purification of inclusion bodies by solubilization and refolding in alkaline-based buffer containing redox agents.A) Semi-solubilization of inclusion bodies in dH2O (pH 12) after 10 min of incubation. L1, the isolated inclusion bodies; L2, purified protein (final product); L3, removing of host cell proteins; L4, remaining insoluble aggregates after final solubilization (each line duplicated). B) Refolding of soluble protein in buffer containing oxidized and reduced glutathione to reform the disulphide bridges of TACH. The refolded protein showed different levels under reducing and non-reducing conditions of the SDS-PAGE, indicating the formation of disulphide bridges. C) SDS-PAGE analysis of the fusion protein. L1, a single band at the expected size for the monomer. L2, double bands of the fusion protein. The upper faint band at the dimer size and the lower thick band at the monomer size (arrows).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139248.g003: Purification of inclusion bodies by solubilization and refolding in alkaline-based buffer containing redox agents.A) Semi-solubilization of inclusion bodies in dH2O (pH 12) after 10 min of incubation. L1, the isolated inclusion bodies; L2, purified protein (final product); L3, removing of host cell proteins; L4, remaining insoluble aggregates after final solubilization (each line duplicated). B) Refolding of soluble protein in buffer containing oxidized and reduced glutathione to reform the disulphide bridges of TACH. The refolded protein showed different levels under reducing and non-reducing conditions of the SDS-PAGE, indicating the formation of disulphide bridges. C) SDS-PAGE analysis of the fusion protein. L1, a single band at the expected size for the monomer. L2, double bands of the fusion protein. The upper faint band at the dimer size and the lower thick band at the monomer size (arrows).
Mentions: To retrieve the bioactivity of the recombinant peptide-fusion protein, pure inclusion bodies were solubilized and refolded in alkaline-based buffer containing redox agents. Although the inclusion bodies were washed extensively, the host cells contaminants were retained after refolding. Therefore, the purified inclusion bodies were washed with dH2O (pH 12) after 10 min of incubation. This step is important to solubilize the host cell proteins that may aggregate with the inclusion bodies; the collected supernatant showed only the host cell contaminants (Fig 3A). The final solubilization of the inclusion bodies was achieved at pH 12.5 in the presence of 2 M urea. The remaining inclusion bodies were insoluble even with higher concentrations of urea, showing that the recombinant peptide-fusion protein was approximately 60% of the total inclusion bodies as shown in Fig 2C and Fig 3A.

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