Strauch Eva-Maria, Georgiou George
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Protein Sci. 2007 May;16(5):1001-8. doi: 10.1110/ps.062687207.
We have developed a bacterial two-hybrid system for the detection of interacting proteins that capitalizes on the folding quality control mechanism of the Twin Arginine Transporter (Tat) pathway. The Tat export pathway is responsible for the membrane translocation of folded proteins, including proteins consisting of more than one polypeptide, only one of which contains a signal peptide ("hitchhiker export"). Here, one protein (bait) is expressed as a fusion to a Tat signal peptide, whereas the second protein (prey) is fused to a protein reporter that can confer a phenotype only after export into the bacterial periplasmic space. Since the prey-reporter fusion lacks a signal peptide, it can only be exported as a complex with the bait-signal peptide fusion that is capable of targeting the Tat translocon. Using maltose-binding protein as a reporter, clones expressing interacting proteins can be grown on maltose minimal media or on MacConkey plates. In addition, we introduce the use of the cysteine disulfide oxidase DsbA as a reporter. Export of a signal peptide-prey:bait-DsbA complex into the periplasm allows complementation of dsbA(-) mutants and restores the formation of active alkaline phosphatase, which in turn can be detected by a chromogenic assay.
我们开发了一种用于检测相互作用蛋白的细菌双杂交系统，该系统利用了双精氨酸转运蛋白（Tat）途径的折叠质量控制机制。Tat输出途径负责折叠蛋白的膜转运，包括由多个多肽组成的蛋白，其中只有一个含有信号肽（“搭便车输出”）。在这里，一种蛋白（诱饵）被表达为与Tat信号肽的融合体，而第二种蛋白（猎物）则与一种蛋白报告基因融合，该报告基因只有在输出到细菌周质空间后才能赋予一种表型。由于猎物-报告基因融合体缺乏信号肽，它只能作为与能够靶向Tat转运体的诱饵-信号肽融合体的复合物输出。使用麦芽糖结合蛋白作为报告基因，表达相互作用蛋白的克隆可以在麦芽糖基本培养基或麦康凯平板上生长。此外，我们引入了半胱氨酸二硫键氧化酶DsbA作为报告基因。信号肽-猎物：诱饵-DsbA复合物输出到周质中可使dsbA（-）突变体互补，并恢复活性碱性磷酸酶的形成，进而可通过显色测定法检测到。
