title: Prokaryotic expression and purification of soluble goldfish Tgf2 transposase with transposition activity.
abstract: Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.
authors.affiliation: College of Food Science and Technology, Shanghai Ocean University, Huchenghuan Road 999, Shanghai, 201306, China.
authors.forename: Hai-Li
authors.initials: HL
authors.lastname: Xu
authors.forename: Xiao-Dan
authors.initials: XD
authors.lastname: Shen
authors.forename: Fei
authors.initials: F
authors.lastname: Hou
authors.forename: Luo-Dan
authors.initials: LD
authors.lastname: Cheng
authors.forename: Shu-Ming
authors.initials: SM
authors.lastname: Zou
authors.forename: Xia-Yun
authors.initials: XY
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029941:Fish Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D006054:Goldfish / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019895:Transposases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D029941:Fish Proteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019895:Transposases