Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1.
Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to obtain sufficient amounts of these receptors for biochemical and structural studies because they often require posttranslational modifications, especially sugar modification. Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni(2+) affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2mg/larva. The sugar profiling revealed that the N-linked sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc and Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc. This revealed that the protein product was much more homogeneous than the complex-sugar type product obtained by mammalian cell expression. The surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary structure. These results clearly suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for their structural and binding studies, due to the relatively homogeneous N-linked sugar modifications.
Sasaki
Kaori
K
Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan.
Kajikawa
Mizuho
M
Kuroki
Kimiko
K
Motohashi
Tomoko
T
Shimojima
Tsukasa
T
Park
Enoch Y
EY
Kondo
Sachiko
S
Yagi
Hirokazu
H
Kato
Koichi
K
Maenaka
Katsumi
K
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D012831:Bombyx / Q000235:genetics*; D002241:Carbohydrates / Q000276:immunology; D004247:DNA / Q000235:genetics; D006458:Hemolymph / Q000276:immunology; D006801:Humans; D054342:Receptors, KIR2DL1 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*
D002241:Carbohydrates; C093100:KIR2DL1 protein, human; D054342:Receptors, KIR2DL1; D011994:Recombinant Proteins; D004247:DNA