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  • 标题:Intein-mediated assembly of a functional β-glucuronidase in transgenic plants
  • 本地全文:下载
  • 作者:Jianjun Yang ; George C. Fox ; Tina V. Henry-Smith
  • 期刊名称:Proceedings of the National Academy of Sciences
  • 印刷版ISSN:0027-8424
  • 电子版ISSN:1091-6490
  • 出版年度:2003
  • 卷号:100
  • 期号:6
  • 页码:3513-3518
  • DOI:10.1073/pnas.0635899100
  • 语种:English
  • 出版社:The National Academy of Sciences of the United States of America
  • 摘要: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.
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