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Expression and Purification of Peanut Oleosins in Insect Cells

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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 k Da 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.

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Abbreviations

TNM-FH:

Trichoplusia Ni medium-formulation hinks

FBS:

Fetal bovine serum

PBS:

Phosphate-buffered solution

DSC:

Differential scanning calorimetry

SDS-PAGE:

Sodium dodecyl sulfate—polyacrylamide gel electrophoresis

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Acknowledgments

We thank Prof. Reiko Urade (Kyoto University) for the warm encouragement. This work was supported in part by grants from A-STEP program of Japan Science and Technology Agency (to N.M.). The first author also wishes to thank the Ministry of Education, Culture, Sports, Science, and Technology of Japan for the scholarship support.

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Authors and Affiliations

  1. Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan

    Cerrone Cabanos, Hiroki Katayama, Shigeru Utsumi & Nobuyuki Maruyama

  2. Phadia KK, Tokyo, Japan

    Akira Tanaka

Authors
  1. Cerrone Cabanos
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  2. Hiroki Katayama
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  3. Akira Tanaka
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  4. Shigeru Utsumi
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  5. Nobuyuki Maruyama
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Corresponding author

Correspondence to Nobuyuki Maruyama.

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Cabanos, C., Katayama, H., Tanaka, A. et al. Expression and Purification of Peanut Oleosins in Insect Cells. Protein J 30, 457–463 (2011). https://doi.org/10.1007/s10930-011-9351-z

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  • DOI: https://doi.org/10.1007/s10930-011-9351-z

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