TY - JOUR
T1 - Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system
AU - Kusaka, Hiroki
AU - Kita, Shunsuke
AU - Tadokoro, Takashi
AU - Yoshida, Kouki
AU - Kasai, Yoshiyuki
AU - Niiyama, Harumi
AU - Fujimoto, Yukari
AU - Hanashima, Shinya
AU - Murata, Michio
AU - Sugiyama, Shigeru
AU - Ose, Toyoyuki
AU - Kuroki, Kimiko
AU - Maenaka, Katsumi
N1 - Funding Information:
This study was supported by the Japanese Society of Technology (JST) ERATO Lipid Active Structure Project JPMJER1005 , JSPS KAKENHI 19K16051 , by “ Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers ” (no. S2701 ), by the Platform Project for Supporting in Drug Discovery and Life Science Research(Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) , Japan Agency for Medical Research and Development (AMED) under Grant Number 16am0101007j0005 , by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number 17am0101093j0001 , 18am0101093j0002 and 19am0101093j0003 , by Hokkaido University , Global Facility Center (GFC) , Pharma Science Open Unit (PSOU) , funded by MEXT under “ Support Program for Implementation of New Equipment Sharing System ”, by Hokkaido University Biosurface project , by Mishima Kaiun Memorial Foundation and by Takeda Science Foundation .
Publisher Copyright:
© 2020 Elsevier Inc.
PY - 2020/8
Y1 - 2020/8
N2 - CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.
AB - CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.
KW - Baculovirus
KW - CD1d
KW - Differential scanning calorimetry
KW - Major histocompatibility complex class I
KW - Secretory expression
KW - Silkworm
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U2 - 10.1016/j.pep.2020.105631
DO - 10.1016/j.pep.2020.105631
M3 - Article
C2 - 32213313
AN - SCOPUS:85082765980
SN - 1046-5928
VL - 172
JO - Protein Expression and Purification
JF - Protein Expression and Purification
M1 - 105631
ER -