title: Functional analysis of the split Synechocystis DnaE intein in plant tissues by biolistic particle bombardment.
abstract: The DnaE intein of Synechocystis sp. PCC6803 (Ssp DnaE intein) is the first split intein identified in nature. Its N-terminal fragment (Int-n) is attached to the end of the N-terminal half of the DnaE protein (DnaE-n) to form the precursor DnaE-n/Int-n, while the C-terminal fragment (Int-c) precedes the C-terminal half of the DnaE protein (DnaE-c) to form the precursor Int-c/DnaE-c. Int-n and Int-c fragments in the separate precursors catalyze, in concert, a protein trans-splicing process to splice the flanking DnaE-n and DnaE-c into a functional catalytic subunit of DNA polymerase III. They then release themselves from the precursors. Previously, the Ssp DnaE intein has been used to reconstitute a protein trans-splicing mechanism in stably transformed Arabidopsis thaliana, resulting in successful reassembly of an intact and functional GUS from two halves of a split GUS protein. In this report, transient expression using a biolistic particle bombardment approach is described for functional analysis of Ssp DnaE intein. Analyses confirmed that the Ssp DnaE intein could catalyze protein trans-splicing not only in model plants but also in monocot and dicot crops. It also demonstrated that when up to 45 amino acid residues were removed from the C-terminus of the Int-n fragment, the Int-n fragment was still able to function in the protein trans-splicing process.
authors.affiliation: Central Research and Development, E. I. DuPont de Nemours & Co., Experimental Station, Wilmington, DE 19880, USA. jianjun-gene.yang@usa.dupont.com
authors.forename: Jianjun
authors.initials: J
authors.lastname: Yang
authors.forename: Tina V
authors.initials: TV
authors.lastname: Henry-Smith
authors.forename: Min
authors.initials: M
authors.lastname: Qi
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000382:microbiology*; D019470:Biolistics; D004258:DNA Polymerase III / Q000235:genetics*; D047668:Inteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion; D046939:Synechocystis / Q000201:enzymology / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III