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.