Prokaryotic Expression and Purification of Soluble Goldfish Tgf2 Transposase with Transposition Activity

• Published in: Molecular biotechnology 2015-01, Vol.57 (1), p.94-100
• Main Authors: Xu, Hai-Li, Shen, Xiao-Dan, Hou, Fei, Cheng, Luo-Dan, Zou, Shu-Ming, Jiang, Xia-Yun
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aquaculture; Biochemistry; Biological Techniques; Biotechnology; Carassius auratus; Cell Biology; Chemistry; Chemistry and Materials Science; chromatography; DNA Transposable Elements; E coli; Electrophoresis, Polyacrylamide Gel; Enzymes; Escherichia coli; Escherichia coli - metabolism; Fish; Fish Proteins - chemistry; Fish Proteins - isolation & purification; Fish Proteins - metabolism; genes; Genetic Vectors - metabolism; goldfish; Goldfish - metabolism; Green Fluorescent Proteins - metabolism; Human Genetics; insertional mutagenesis; Low temperature; Mass spectrometry; Megalobrama amblycephala; Molecular Sequence Data; molecular weight; plasmids; Protein expression; Protein Science; protein structure; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Solubility; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transposases - chemistry; Transposases - isolation & purification; Transposases - metabolism; transposition (genetics); transposons; trypsin
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-014-9805-6

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 Ni²⁺-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.