Chen L, Pradhan S, Evans T C
New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Gene. 2001 Jan 24;263(1-2):39-48. doi: 10.1016/s0378-1119(00)00568-0.
We report that the N- and C-terminal splicing domains of the intein found in the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) are capable of association in vivo and in vitro, even with key splicing residues changed to alanine (Cys(1), Asn(159), and Cys(+1) to Ala). These studies utilized the herbicide resistant form of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Salmonella typhimurium and an Escherichia coli strain with the EPSPS gene deleted from its genome (E. coli strain ER2799). EPSPS was mapped to identify potential split sites using a facile Tn7 linker scanning procedure. Forty positions were found to tolerate a five amino acid insertion while 21 sites did not, as assayed by the rescue of growth of E. coli strain ER2799. Further characterization of these sites by inserting a full length Ssp DnaE intein identified residue 235 of EPSPS as the optimal position. The EPSPS gene was then divided into amino acids 1-235 and 236-427 which were fused to residues 1-123 and 124-159 of a splicing defective Ssp DnaE intein, respectively. Expression of the EPSPS-intein fusions from separate DNA molecules conferred resistance to the herbicide glyphosate, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. As a control the split EPSPS without the intein-affinity domain did not allow cell growth. The use of an intein as an in vivo affinity domain was termed intein-mediated protein complementation (IPC). Intein fragment assembly was verified in vitro by immobilizing the C-terminal splicing domain of the Ssp DnaE intein on a resin and demonstrating that the N-terminal 235 amino acids of EPSPS only bound to the resin when fused to the N-terminal splicing domain of the Ssp DnaE intein. As chloroplast DNA is not transmitted by pollen in plants such as corn and soybean, transgene spread via pollen may be controlled in the future by expressing inactive gene fragments from separate DNA locations, such as the nuclear and chloroplast genome, and using the split intein to generate protein activity.
我们报道，集胞藻属PCC6803（Ssp DnaE内含肽）的dnaE基因中发现的内含肽的N端和C端剪接结构域在体内和体外均能发生关联，即使关键剪接残基被替换为丙氨酸（Cys(1)、Asn(159)和Cys(+1)替换为Ala）。这些研究利用了鼠伤寒沙门氏菌中抗除草剂形式的5-烯醇丙酮酸莽草酸-3-磷酸合酶（EPSPS）以及基因组中缺失EPSPS基因的大肠杆菌菌株（大肠杆菌菌株ER2799）。使用简便的Tn7接头扫描程序对EPSPS进行定位以确定潜在的切割位点。通过检测大肠杆菌菌株ER2799的生长恢复情况发现，40个位置可耐受五个氨基酸的插入，而21个位点则不能。通过插入全长Ssp DnaE内含肽对这些位点进行进一步表征，确定EPSPS的第235位残基为最佳位置。然后将EPSPS基因分为1-235位氨基酸和236-427位氨基酸，它们分别与剪接缺陷型Ssp DnaE内含肽的1-123位残基和124-159位残基融合。来自单独DNA分子的EPSPS-内含肽融合体的表达赋予了对除草剂草甘膦的抗性，这表明内含肽剪接结构域将EPSPS片段聚集在一起以产生活性。作为对照，没有内含肽亲和结构域的切割后的EPSPS不能使细胞生长。将内含肽用作体内亲和结构域被称为内含肽介导的蛋白质互补（IPC）。通过将Ssp DnaE内含肽的C端剪接结构域固定在树脂上，并证明只有当EPSPS的N端235个氨基酸与Ssp DnaE内含肽 的N端剪接结构域融合时才会与树脂结合，从而在体外验证了内含肽片段的组装。由于在玉米和大豆等植物中叶绿体DNA不会通过花粉传播，未来通过从单独的DNA位置（如核基因组和叶绿体基因组）表达无活性的基因片段，并使用切割后的内含肽产生蛋白质活性，可能会控制转基因通过花粉传播。
