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 InsP6 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 nonorganic solvent. The final yield of purified rPLP was 36%. The Kd and Bmax values for the InsP6-PLP binding were 55 nM and 33 pmol/microgram protein, respectively. The Kd value of purified rPLP is equal to that of mouse brain PLP. These results indicate that purified rPLP keeps its native conformation and binds InsP6 in an almost one-to-one ratio.
Advanced Research Laboratory, Hitachi Ltd., Saitama, Japan. fukuzono@cm.naka.hitachi.co.jp
S
S
Fukuzono
T
T
Takeshita
T
T
Sakamoto
A
A
Hisada
N
N
Shimizu
K
K
Mikoshiba
D000368:Aged; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D006801:Humans; D007163:Immunosorbent Techniques; D051379:Mice; D018991:Myelin Proteolipid Protein / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D010833:Phytic Acid / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D018991:Myelin Proteolipid Protein; D011994:Recombinant Proteins; D010833:Phytic Acid