title: Engineering of a novel tri-functional enzyme with MnSOD, catalase and cell-permeable activities.
abstract: Cooperative function of superoxide dismutase (SOD) and catalase (CAT), in protection against oxidative stress, is known to be more effective than the action of either single enzyme. Chemical conjugation of the two enzymes resulted in molecules with higher antioxidant activity and therapeutic efficacy. However, chemical methods holds several drawbacks; e.g., loss of enzymatic activity, low homogeneity, time-consuming, and the need of chemical residues removal. Yet, the conjugated enzymes have never been proven to internalize into target cells. In this study, by employing genetic and protein engineering technologies, we reported designing and production of a bi-functional protein with SOD and CAT activities for the first time. To enable cellular internalization, cell penetrating peptide from HIV-1 Tat (TAT) was incorporated. Co-expression of CAT-MnSOD and MnSOD-TAT fusion genes allowed simultaneous self-assembly of the protein sequences into a large protein complex, which is expected to contained one tetrameric structure of CAT, four tetrameric structures of MnSOD and twelve units of TAT. The protein showed cellular internalization and superior protection against paraquat-induced cell death as compared to either complex bi-functional protein without TAT or to native enzymes fused with TAT. This study not only provided an alternative strategy to produce multifunctional protein complex, but also gained an insight into the development of therapeutic agent against oxidative stress-related conditions.
authors.affiliation: Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Piriya
authors.initials: P
authors.lastname: Luangwattananun
authors.affiliation: Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand; Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Electronic address: sakda.yai@mahidol.ac.th.
authors.forename: Sakda
authors.initials: S
authors.lastname: Yainoy
authors.affiliation: Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand; Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Warawan
authors.initials: W
authors.lastname: Eiamphungporn
authors.affiliation: Department of Community Medical Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand; Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Napat
authors.initials: N
authors.lastname: Songtawee
authors.affiliation: Pure and Applied Biochemistry, Department of Chemistry, Lund University, SE-221 00 Lund, Sweden.
authors.forename: Leif
authors.initials: L
authors.lastname: Bülow
authors.affiliation: Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Chartchalerm Isarankura Na
authors.initials: CI
authors.lastname: Ayudhya
authors.affiliation: Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Electronic address: virapong.pra@mahidol.ac.th.
authors.forename: Virapong
authors.initials: V
authors.lastname: Prachayasittikul
mesh_terms: D002374:Catalase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002850:Chromatography, Gel; D004789:Enzyme Activation; D006418:Heme / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D006418:Heme; D002374:Catalase; D013482:Superoxide Dismutase
keywords: Catalase; Fusion protein; HIV-1 TAT; MnSOD; Oxidative stress; Protein engineering