Functional analysis of the split Synechocystis DnaE intein in plant tissues by biolistic particle bombardment.
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.
Yang
Jianjun
J
Central Research and Development, E. I. DuPont de Nemours & Co., Experimental Station, Wilmington, DE 19880, USA. jianjun-gene.yang@usa.dupont.com
Henry-Smith
Tina V
TV
Qi
Min
M
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*
D011993:Recombinant Fusion Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III