Expression Of Functional Domains Of LRRK2in Escherichia Coli And Sf21Cells

Parkinson’s Disease (PD, OMIM no.168600) is the most common motorneurodegenerative disorder. LRRK2has been one of the familial parkinsonism-relatedgenes. It has2527amino acids and contains5conserved domains(leucine-rich-repeat,Roc GTPase, C terminal of Roc, kinaseand WD40domains) at the C-terminal.Despite intensive study, knowledge of crystal strucure and functions of LRRK2remains far from known. This study aimed to express the recombinant functionaldomains of LRRK2, which can further be used to study the crystal structure and theunderlying mechanism in LRRK2-related physiology and pathology. A predictedfunctional fragment of LRRK2containing the LRR, Roc, COR, kinaseand WD40domains (we named it LLC) was expressed in E. coliusing2different vectors,pET-32b and pE-SUMOstar, respectively. However, such expressed recombinantprotein formed inclusion bodies. To promote the solubility and obtain the activeprotein, BaculoDirectTMbaculovirus expression system and Sf21insect cells wereadopted for expression of LLC fragment with the SUMOstar tag. Using the TOPO cloning technology, we first cloned the LLC gene into thepENTRTM/TEV/D-TOPO vector, then theGateway LR recombination technology was employed to constructthe recombinant bacmid, which was subsequently used for transfection of Sf21insectcells directly.Western Blot was performed to detect the expression of LLC fragment.LLC fragment was successfully expressed in the cells transfected with the P1baculovirus. At the same time, we found that the protein of interest may alsodegradeto a certain extend, indicating some measures should be considered toimprove the stability in the following work. To sum, this study has tried differentprotein expression system and explored the methods using BaculoDirectTMbaculovirus expression system for expression of the functional domains of LRRK2,which has laid a foundation for the research of structure and functions of LRRK2.