Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield.
Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.
Zhang
Yuansong
Y
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan. zyshjh@hotmail.com
Hu
Junhua
J
Miao
Yungen
Y
Zhao
Aichun
A
Zhao
Tianfu
T
Wu
Dayang
D
Liang
Liefeng
L
Miikura
Ayumi
A
Shiomi
Kunihiro
K
Kajiura
Zenta
Z
Nakagaki
Masao
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005349:Fibroins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013112:Spiders / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005349:Fibroins