Funded Studies
Study Rationale:
Mutations in the gene that encodes the LRRK2 protein are frequent causes of Parkinson’s disease (PD). An obstacle to understanding how these mutations lead to PD is the lack of understanding of LRRK2 function and its activation mechanism. Biochemical and structural characterization of LRRK2 will reveal its function, but they require functional LRRK2 protein. However, production of this protein is challenging and almost all attempts to express and purify LRRK2 have failed. We are using a directed evolution approach to increase the expression of functional LRRK2 protein.
Hypothesis:
By directed evolution of bacteria, we can obtain better protein expression hosts that are required for LRRK2 production. Thereby, adequate amounts of functional LRRK2 will become available for structural, biochemical and pharmaceutical investigations.
Study Design:
The LRRK2 gene is fused with the genes that encode green fluorescence protein (GFP), a folding reporter, and ermC, erythromycin resistance protein. The resulting fusion protein will be expressed in bacteria, and a stepwise increase of erythromycin will drive increase in expression of LRRK2 fusion protein. GFP is used to select for LRRK2 in a folded state. The evolved bacteria will then be used for expressing and purifying LRRK2.
Impact on Diagnosis/Treatment of Parkinson’s Disease:
Our proposed project aims to overcome the bottleneck of studying the structure and function of LRRK2. This is critical for understanding LRRK2-mediated PD and developing targeted therapeutics.
Next Steps for Development:
The evolved bacteria will be used as the expression hosts for LRRK2 protein production, which can be used for pharmaceutical studies aiming at development of drugs for clinical use.