title: Intein-mediated assembly of a functional beta-glucuronidase in transgenic plants.
abstract: The DnaE intein in Synechocystis sp. strain PCC6803 is the first and only naturally split intein that has been identified so far. It is capable of catalyzing a protein trans-splicing mechanism to assemble a mature protein from two separate precursors. Therefore, it is a powerful tool for protein modification and engineering. Inteins have not been identified, nor have intein-mediated protein splicing reactions been demonstrated, in plant cells. In this paper, we describe the use of the Ssp DnaE split intein in transgenic plants for reconstitution of a protein trans-splicing reaction. We have synthesized artificial genes that encode for N-terminal half (Int-n) and C-terminal half (Int-c) fragments of Ssp DnaE split intein and divided beta-glucuronidase (GUS) gene to encode GUS-n and GUS-c parts of the enzyme as reporter. The in-frame fusions of GUSn/Intn and Intc/GUSc were constructed and transfected into Arabidopsis. We have observed in vivo reassembly of functional beta-glucuronidase when both GUSn/Intn and Intc/GUSc constructs were introduced into the same Arabidopsis genome either by cotransformation or through genetic crossing, hereby signifying an intein-mediated protein trans-splicing mechanism reconstituted in plant cells.
authors.affiliation: Central Research and Development, E. I. DuPont de Nemours & Co. Experimental Station, Wilmington, DE 19880-0402, USA. Jianjun-Gene.Yang@usa.dupont.com
authors.forename: Jianjun
authors.initials: J
authors.lastname: Yang
authors.forename: George C
authors.initials: GC
authors.lastname: Fox
authors.forename: Tina V
authors.initials: TV
authors.lastname: Henry-Smith
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003433:Crosses, Genetic; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D019154:Protein Splicing; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D005966:Glucuronidase