Intein-mediated assembly of a functional beta-glucuronidase in transgenic plants.
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.
Yang
Jianjun
J
Central Research and Development, E. I. DuPont de Nemours & Co. Experimental Station, Wilmington, DE 19880-0402, USA. Jianjun-Gene.Yang@usa.dupont.com
Fox
George C
GC
Henry-Smith
Tina V
TV
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
D004274:DNA, Recombinant; D011994:Recombinant Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D005966:Glucuronidase