Yang Jianjun, Fox George C, Henry-Smith Tina V
Central Research and Development, E. I. DuPont de Nemours & Co. Experimental Station, Wilmington, DE 19880-0402, USA.
Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3513-8. doi: 10.1073/pnas.0635899100. Epub 2003 Mar 10.
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.
集胞藻6803株中的DnaE内含肽是目前已鉴定出的首个也是唯一的天然分裂内含肽。它能够催化蛋白质反式剪接机制，从两个独立的前体组装成一个成熟的蛋白质。因此，它是蛋白质修饰和工程的有力工具。在植物细胞中尚未鉴定出内含肽，也未证明存在内含肽介导的蛋白质剪接反应。在本文中，我们描述了在转基因植物中使用Ssp DnaE分裂内含肽来重建蛋白质反式剪接反应。我们合成了编码Ssp DnaE分裂内含肽N端半段（Int-n）和C端半段（Int-c）片段的人工基因，并将β-葡萄糖醛酸酶（GUS）基因分割，以编码该酶的GUS-n和GUS-c部分作为报告基因。构建了GUSn/Intn和Intc/GUSc的读码框融合体并转染到拟南芥中。当通过共转化或遗传杂交将GUSn/Intn和Intc/GUSc构建体都导入同一拟南芥基因组时，我们观察到了功能性β-葡萄糖醛酸酶的体内重新组装，从而表明在植物细胞中重建了内含肽介导的蛋白质反式剪接机制。
