Overproduction and Immuno-affinity Purification of Myelin Proteolipid Protein (PLP), an Inositol Hexakisphosphate-Binding Protein, in a Baculovirus Expression System
| Autor: | Shinichi Fukuzono, Akiko Hisada, Katsuhiko Mikoshiba, Tomoko Takeshita, Norio Shimizu, Takeshi Sakamoto |
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| Rok vydání: | 1998 |
| Předmět: |
Phytic Acid
Inositol hexakisphosphate binding Biophysics Biology Biochemistry Chromatography Affinity law.invention Mice Myelin Affinity chromatography immune system diseases law Native state medicine Animals Humans Myelin Proteolipid Protein Overproduction Molecular Biology Integral membrane protein Immunosorbent Techniques Aged Cell Biology Molecular biology Recombinant Proteins nervous system diseases Myelin proteolipid protein medicine.anatomical_structure Recombinant DNA lipids (amino acids peptides and proteins) Baculoviridae Protein Binding |
| Zdroj: | Biochemical and Biophysical Research Communications. 249:66-72 |
| ISSN: | 0006-291X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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