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Intein-Mediated Assembly of a Functional β-Glucuronidase in Transgenic Plants
Jianjun Yang, George C. Fox, Jr. and Tina V. Henry-Smith
Proceedings of the National Academy of Sciences of the United States of America
Vol. 100, No. 6 (Mar. 18, 2003), pp. 3513-3518
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3139391
Page Count: 6
You can always find the topics here!Topics: Proteins, Trans splicing, Splicing, Hybridity, Amino acids, Seedlings, Plant cells, Plants, Plasmids, Transgenic plants
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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 β-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 β-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.
Proceedings of the National Academy of Sciences of the United States of America © 2003 National Academy of Sciences