Hyperproduction of Polyhedrin-IGF II Fusion Protein in Silkworm Larvae Infected with Recombinant Bombyx mori Nuclear Polyhedrosis Virus

Abstract

SUMMARYA gene coding for insulin-like growth factor II (IGF II) was constructed from 16 oligodeoxynucleotides synthesized chemically and cloned into EcoRI-SalI sites of pBR322. In this gene an ATG codon for methionine was introduced for cleavage by CNBr at the beginning of mature IGF II. For expressing foreign genes, a new hostvector system, with Bombyx mori silkworm larvae as the host and B. mori nuclear polyhedrosis virus (BmNPV) as the vector, has been developed. BmNPV genomic DNA codes polyhedrin which is a major protein of inclusion bodies and is massproduced in infected silkworm larvae. We employed this polyhedrin production system to obtain a large yield of a foreign gene product. The coding region of the carboxyterminal half of polyhedrin was removed and the remainder was ligated with the IGF II gene in phase to create a fusion protein gene consisting of the coding region of the amino-terminal half of polyhedrin and the IGF II gene. This fusion protein gene was combined in a plasmid with the promoter and 5' and 3' flanking regions of the polyhedrin gene. The resulting plasmid and the wild-type BmNPV genomic DNA were cotransfected into BM-N cells, and a recombinant virus was isolated by the limiting dilution method. The silkworm larvae infected with the recombinant virus produced 3-6 mg of the fusion protein per larva and the infected BM-N cells produced 0-3 mg per ml of culture. IGF II was released from the fusion protein produced by BM-N cells infected with the recombinant virus by CNBr treatment, purified by extraction with guanidine-HC1, column chromatography and HPLC and the correct amino-terminal amino acid sequence confirmed. INTRODUCTIONIn order to express foreign genes, new host vector systems employing insect viruses and insect cell lines or larval bodies have recently been developed. Recombinant vaccinia virus was created to produce Sindbis virus proteins in Aedes albopictus mosquito cell lines (Franke & Hruby, 1985).

Binding Sites and Binding Properties of Binary and Ternary Complexes of Insulin-like Growth Factor-II (IGF-II), IGF-binding Protein-3, and Acid-labile Subunit

“…In this study, the results of the IGFs/IGFBP-3/ALS solid-phase binding assay suggest that mutation of Tyr 27 to Leu does not support ternary complex formation. Similar results have been obtained by other groups using IGF-I mutants in which Tyr 24 substituted with Leu ([Leu 24 ]rIGF-I) reduced the ternary complex formation activity (21). Tyr 24 of IGF-I is also important for binding to insulin and IGF-I receptors (35).…”

“…A double mutant of [Leu 27 , Leu 43 ]rIGF-II was constructed by identical procedures using synthetic oligonucleotides. Oligonucleotides were synthesized by an Applied Biosystems model 380A synthesizer, purified as described previously (24), and sequences were confirmed by the dideoxynucleotide chain termination method (25) using a 7-DEAZA sequencing kit (Takara Shuzo, Kyoto, Japan).…”

“…These natural fragments corresponding to the amino-terminal domain of IGFBP-3 were isolated without disulfide reduction, suggesting that these domains are not linked by disulfide bonds (6). In the case of the IGFs/IGFBP-3/ALS ternary complex formation, Tyr 24 and the D-domain (residues 63-70) of human IGF-I are thought to be important (21). However, the properties of IGFBP-3 and ALS for ternary complex formation are not understood well.…”

Hyper-Enhanced Production of Foreign Recombinant Protein by Fusion with the Partial Polyhedrin of Nucleopolyhedrovirus

“…As a result of ongoing studies and efforts over the last decade, BEVS has evolved to overcome some of these technical issues [11], [12]. Many researchers have performed studies to resolve this limitation, including the alteration of promoter sequences, fusion expression with partial polyhedrin or various tagging signals and co-expression with regulatory proteins[13]–[18]. Although these techniques could enhance the expression efficiency somewhat, they were not entirely satisfactory.…”

Purification and Refolding of Recombinant Human IGF II from Silkworms Infected with RecombinantBombyx moriNuclear Polyhedrosis Virus

“…We have reported the high production of insulin-like growth factor II (IGF II) as the fusion protein in infected silkworm cultured cells and larvae. 10) Methionine was introduced into the fusion protein at the beginning of IGF II, so that IGF II could be released by CNBr treatment. Recently, an attempt to express human IGF II cDNA in insect cells was reported to have resulted in a failure to secrete the product.…”