Overproduction and immuno-affinity purification of myelin proteolipid protein (PLP), an inositol hexakisphosphate-binding protein, in a baculovirus expression system

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 baculo-virus 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 nonorganic 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/mu g 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. (C) 1998 Academic Press.