Overproduction and Immuno-affinity Purification of Myelin Proteolipid Protein (PLP), an Inositol Hexakisphosphate-Binding Protein, in a Baculovirus Expression System

Abstract Myelin proteolipid protein (PLP) is a major integral membrane protein of central nervous system myelin and is considered to play a significant role in myelination. PLP has a four-transmembrane structure, judging from the hydropathy profile. In addition, it has InsP 6 binding activity. Here, we have succeeded in producing PLP in large quantities of 3.9 pg/cell (6 mg/L) by using a baculovirus expression system and developing an efficient purification method, maintaining InsP6 binding activity. The recombinant PLP (rPLP) was purified by ion-exchange and immunoaffinity chromatography in a non-organic solvent. The final yield of purified rPLP was 36%. The K d and B max values for the InsP 6 -PLP binding were 55 nM and 33 pmol/μg protein, respectively. The K d value of purified rPLP is equal to that of mouse brain PLP. These results indicate that purified rPLP keeps its native conformation and binds InsP 6 in an almost one-to-one ratio.