Expression, purification and preliminary biochemical studies of the N-terminal domain of leucine-rich repeat kinase 2.
Leucine-rich repeat kinase 2 gene is a key factor for Parkinson's disease and encodes for a large protein kinase LRRK2 (280kDa) with multiple domains, including the different repeat sequences at the N-terminus such as ankyrin domain. Here, we successfully expressed and purified two kinds of LRRK2's N-terminal fragments N1 (aa12-320) and N2 (aa12-860). The purified N2 protein was identified by mass spectrometry and N1's molecular weight was determined to be 33.23kDa. Gel filtration revealed that N1 exhibits as monomer, dimer and tetramer and N2 as oligomer in solution. N1's multiple oligomeric states were further proved by native-page and cross-linking gel experiments. Circular dichroism spectrum indicated that N1 and N2 contain both alpha helixes and beta sheets. The polymerization character of LRRK2 N-terminal region would be speculated to relate with its biological function.
Lu
Bin
B
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Zhai
Yujia
Y
Wu
Chan
C
Pang
Xiaoyun
X
Xu
Zhiheng
Z
Sun
Fei
F
D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D013058:Mass Spectrometry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D003432:Cross-Linking Reagents; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases