A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.
A silk thread of the silkworm, Bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior and middle silk gland (MSG), respectively. This study aimed to develop a novel sericin 1 gene (ser1) promoter-driven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized in MSG and secreted into the sericin layer. To obtain a high level of gene expression, we tested whether a baculovirus-derived enhancer, hr3, and a trans-regulator, IE1, are capable of stimulating the transcriptional activity of the ser1 promoter, using a transient gene expression system. The results showed that hr3 and IE1 cooperatively increased the ser1 promoter activity more than 30-fold. Then, transgenic silkworms were generated which expressed the EGFP with the signal peptide in MSG under the control of the hr3-linked ser1 promoter and IE1 gene. The silkworms exclusively secreted the EGFP into the sericin layer of cocoons as predicted. The expressed EGFP was extractable from cocoons through a simple procedure with neutral pH buffer solution. The expression system developed in this study enables us to produce recombinant proteins in bulk that can be easily extracted and purified.
Tomita
Masahiro
M
Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima, 739-0046, Japan.
Hino
Rika
R
Ogawa
Shingo
S
Iizuka
Masashi
M
Adachi
Takahiro
T
Shimizu
Katsuhiko
K
Sotoshiro
Hisaya
H
Yoshizato
Katsutoshi
K
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D047030:Sericins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047011:Silk / Q000737:chemistry*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D047030:Sericins; D047011:Silk; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases