LAMR1与PER1蛋白作用位点的筛选

Screening for Key Region of Laminin Receptor 1 Interacted with PERIOD 1

目的:筛选出与PER1蛋白相互作用的LAMR1的核心位点。方法:采用盒式定点突变法,对重组目的质粒pGADT7-Rec/Lamr1201-295的插入片段Lamr1201-295羧基末端的205-216氨基酸序列RDPEEIEKEEQA进行定点突变,并以hPER1蛋白的bHLH-PAS结构域为诱饵蛋白,通过酵母双杂交系统确定这4种不同的突变LAMR1201-295片段与hPER1的相互作用。结果:营养缺陷培养基筛选显示4种转化菌在二缺SD/-Leu/-Trp、三缺SD/-His/-Leu/-Trp和四缺SD/-Ade/-His/-Leu/-Trp固体培养基上都可以生长;β-半乳糖苷酶印膜法检测结果显示4种转化菌均可显色。结论:突变片段LAMR1-RDP,LAMR1-EEI,LAMR1-EKE和LAMR1-EQA都能够与hPER1的bHLH-PAS结构域在酵母中发生相互作用。提示LAMR1的205-216Aa可能不是其与hPER1相互作用的核心氨基酸序列。

Objective： To define the key region of LAMR1 to interact with hPER1. Methods： With cassette mutagenesis, pGADT7-Rec/hLamr1201-295 was mutated, targeting the sequence RDP, EEI, EKE and EQA at carboxyl terminal, respectively. And the interaction between PER1 and four kinds of mutated LAMR1201-295 were further identified using a yeast two-hybrid system. Results： Clones containing bHLH-PAS domain of Per1 and Lamrl could be seen in SD/-Leu/-Trp, SD/-His/-Leu/-Trp and SD-Ade/-His/-Leu/-Trp selection mediums. The blue stain was also obviously observed in colony-lift filter 13-galactosidase assay. Conclusions： hPER1 could interact with all mutant LAMR1, which suggested that the sequence RDPEEIEKEEQA at carboxyl terminal is not necessary for the interaction to occur.