title: Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics.
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.
authors.affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Hussin A
authors.initials: HA
authors.lastname: Rothan
authors.affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Jamunaa
authors.initials: J
authors.lastname: Ambikabothy
authors.affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Ammar Y
authors.initials: AY
authors.lastname: Abdulrahman
authors.affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Hirbod
authors.initials: H
authors.lastname: Bahrani
authors.affiliation: Department of Medicine, Faculty of Medicine, Medical Centre of University Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia.
authors.forename: Mojtaba
authors.initials: M
authors.lastname: Golpich
authors.affiliation: Department of Medicine, Faculty of Medicine, Medical Centre of University Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia.
authors.forename: Elham
authors.initials: E
authors.lastname: Amini
authors.affiliation: Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Noorsaadah
authors.initials: N
authors.lastname: A Rahman
authors.affiliation: Institute of biological sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Teow Chong
authors.initials: TC
authors.lastname: Teoh
authors.affiliation: Institute of biological sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Zulqarnain
authors.initials: Z
authors.lastname: Mohamed
authors.affiliation: Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Rohana
authors.initials: R
authors.lastname: Yusof
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D004357:Drug Synergism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D015151:Immunoblotting; D002479:Inclusion Bodies / Q000378:metabolism; D061986:MCF-7 Cells; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D054790:Ribosome Inactivating Proteins, Type 2 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C066347:MAP30 protein, Momordica charantia; D010455:Peptides; D011993:Recombinant Fusion Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D004317:Doxorubicin