Screening of LRRK2 interactants by yeast 2-hybrid analysis / 中南大学学报(医学版)

ABSTRACT

Objective To isolate and identify the potential binding partners of LRRK2,a gene linked to both dominant familial form and sporadic form of Parkinson's disease,thus to further our knowledge of its function.Methods We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait.The bait amplified by polymerase chain reaction(PCR) was then cloned into a yeast expression plasmid pGBKT7.After being sequenced and analyzed,pGBKT7-bait was transformed into the yeast strain AH109.Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain.Then human fetal brain cDNA library was trarnsformed into that yeast strain.which could express pGBKT7-bait fusion protein.The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade)containing X-a-gal.We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7,respectively.At last,these plasmids isolated from these true positive colonies were analyzed by bioinformatics.Results We obtained 9 true positive colonies,these colonies were sequenced, and we performed sequence Blast in GenBank.Three colonies of the 9 positive colonies were not in open reading-frames.Among other 6 colonies,there were known proteins including spermatid perinuclear RNA-binding protein(STRBP)and BCL2-associated athanogene 5 isoform b(BAG5),as well as unknown proteins including tyrosine phosphatase non-receptor type(PTPN23),1(3)mbt-like 3 isoform b(L3 MBTL3),RALY RNA binding protein-like isoform 1(RALYL),and Homo sapiens mRNA for KIAA1783 protein,partial cds(KIAA 1783).Conclusion True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid.Our screened proteins may provide a new research clue for revealing biological functions of LRRK2,pathogenesis of Parkinson's disease,and other neurodegerations.