title: A novel multigene cloning method for the production of a motile ATPase.
abstract: With the advent of nanotechnology, new functional modules (e.g., nanomotors, nanoprobes) have become essential in several medical fields. Generally, mechanical modulators systems are the principal components of most cutting-edge technologies in modern biomedical applications. However, the in vivo use of motile probes has raised many concerns due to their low sensitivity and non-biocompatibility. As an alternative, biological enzymatic engines have received increased attention. In particular, ATPases, which belong to a class of motile enzymes that catalyze chemical metabolic reactions, have emerged as a promising motor due to their improved biocompatibility and performance. However, ATPases usually suffer from lower functional activity and are difficult to express recombinantly in bacteria relative to their conventional and synthetic competitors. Here, we report a novel functional modified ATPase with both a simple purification protocol and enhanced motile activity. For this mutant ATPase, a new bacterial subcloning method was established. The ATPase-encoding sequence was redesigned so that the mutant ATPase could be easily produced in an Escherichia coli system. The modified thermophilic F1-ATPase (mTF1-ATPase) demonstrated 17.8unit/mg ATPase activity. We propose that derivatives of our ATPase may enable the development of novel in vitro and in vivo synthetic medical diagnostics, as well as therapeutics.
authors.affiliation: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea. Electronic address: minsujang@skku.edu.
authors.forename: Min Su
authors.initials: MS
authors.lastname: Jang
authors.affiliation: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea. Electronic address: swc1234@skku.edu.
authors.forename: Woo Chul
authors.initials: WC
authors.lastname: Song
authors.affiliation: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea. Electronic address: seung3885@skku.edu.
authors.forename: Seung Won
authors.initials: SW
authors.lastname: Shin
authors.affiliation: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea. Electronic address: kspcharlie@skku.edu.
authors.forename: Kyung Soo
authors.initials: KS
authors.lastname: Park
authors.affiliation: Center for Bionics/Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 136-791, South Korea. Electronic address: jinseok@kist.re.kr.
authors.forename: Jinseok
authors.initials: J
authors.lastname: Kim
authors.affiliation: Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, South Korea. Electronic address: dikim@skku.edu.
authors.forename: Dong-Ik
authors.initials: DI
authors.lastname: Kim
authors.affiliation: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea. Electronic address: bwkim@skku.edu.
authors.forename: Byung Woo
authors.initials: BW
authors.lastname: Kim
authors.affiliation: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, South Korea; SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746, South Korea. Electronic address: sh.um@skku.edu.
authors.forename: Soong Ho
authors.initials: SH
authors.lastname: Um
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases
keywords: Membrane protein; Multi-gene; Self-ligation; Subcloning; mTF(1)-ATPase