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

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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 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.

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Acknowledgments

This work was supported by Grants from the National High Technology Research and Development Program of China (863 Program) (2011AA100403) and the National Science Foundation of China (Nos. 31201760 and 31272633).

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Authors and Affiliations

  1. College of Food Science and Technology, Shanghai Ocean University, Huchenghuan Road 999, Shanghai, 201306, China

    Hai-Li Xu, Fei Hou & Xia-Yun Jiang

  2. Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai, 201306, China

    Xiao-Dan Shen, Luo-Dan Cheng & Shu-Ming Zou

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  1. Hai-Li Xu
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  2. Xiao-Dan Shen
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  3. Fei Hou
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Correspondence to Shu-Ming Zou or Xia-Yun Jiang.

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Hai-Li Xu and Xiao-Dan Shen have contributed equally to this work.

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Xu, HL., Shen, XD., Hou, F. et al. Prokaryotic Expression and Purification of Soluble Goldfish Tgf2 Transposase with Transposition Activity. Mol Biotechnol 57, 94–100 (2015). https://doi.org/10.1007/s12033-014-9805-6

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