title: Expression and purification of peanut oleosins in insect cells.
abstract: Oleosins contain a unique hydrophobic domain which is inserted into the oil matrix and are involved in the formation and stability of plant oil bodies. These proteins have also been reported to possess some allergenic properties. Therefore, knowledge of its three-dimensional structure is vital for further structural and immunological characterization. However, due to the difficulty of soluble recombinant expression in Escherichia coli, no studies have been done in line with this goal. Here, we have developed a novel expression and purification system for three peanut oleosin isoforms (14 k, 16 k, and 18 kDa oleosins). Oleosin cDNAs were cloned and subsequently expressed in soluble form in insect cell-baculovirus system. Recombinant proteins can be purified to homogeneity using only Ni Sepharose affinity chromatography. Thermal denaturation midpoint temperatures of recombinant oleosins were also assayed and found to be very similar to that of native oleosins, indicating proper structural conformation of the recombinant proteins.
authors.affiliation: Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Cerrone
authors.initials: C
authors.lastname: Cabanos
authors.forename: Hiroki
authors.initials: H
authors.lastname: Katayama
authors.forename: Akira
authors.initials: A
authors.lastname: Tanaka
authors.forename: Shigeru
authors.initials: S
authors.lastname: Utsumi
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Maruyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D010367:Arachis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins