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Analysis of Protein Interaction Between S-periaxin by Bimolecular Fluorescence Complementation Assay
Ren Yemei, Shi Yawei*
Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, China; Institute of Biotechnology, Shanxi University, Taiyuan 030006, China
Abstract: Periaxin is expressed by myelinating Schwann cells and plays an essential role in stabilizing the Schwann cell-axon unit in the myelinated fibers of the vertebra. Mutations in the periaxin gene are known to cause autosomal recessive demyelinating Charcot-Marie-Tooth (CMT4F). Periaxin gene encodes L-periaxin and a truncated isoform， S-periaxin， which have an N-terminal PDZ domain and are targeted differently in the Schwann cell. The molecular structure and biological function of S-periaxin are unknown to date. In this work， the DNA sequence encoding S-periaxin was cloned into the vector pET-M-3C to form a recombinant plasmid pET-M-3C-S-periaxin. The recombinant protein was overexpressed in E.coli BL21 and purified by Ni-NTA column and Sephacryl S-200 column. S-periaxin was easy to form the different degree of polymerization in vitro by glutaraldehyde crosslinking. S-periaxin could homodimerize with coimmunoprecipitation. In addition， bimolecular fluorescence complementation (BiFC) system was developed by splitting mCherry. BiFC demonstrated S-periaxin could also form homodimer in E.coli BL21 and RSC96 cell.
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