Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms

Abstract

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

“…Silkworms can be efficiently transformed with piggyBac vectors (25,26), and piggyBac vectors can be engineered to target foreign gene expression in a silk gland-specific fashion (27)(28)(29) and to direct the incor- poration of foreign proteins into composite silk fibers (27)(28)(29)(30)32). The use of piggyBac vectors with these features has yielded transgenic silkworm lines that produced composite fibers containing a variety of different recombinant proteins, including procollagen (28), GFP (27,29), and feline IFN (30). In addition, while our work was in progress, others isolated transgenic silkworms that can produce spider silk proteins (35,36).…”

“…Although silkworms are not recognized as a major recombinant protein production platform (21)(22)(23), they might be the perfect host for spider silk fiber production because transgenic silkworms can be efficiently produced using piggyBac vectors (24)(25)(26), recombinant protein production can be targeted to the silk gland with tissuespecific promoters (27)(28)(29)(30)(31)(32), and the silk gland is naturally equipped to assemble silk proteins into fibers (33). One caveat is that silkworms produce endogenous silk proteins.…”

A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm

“…The transgenic technique has been used in the functional analysis of genes 10,11) and as a tool in foreign gene production. [12][13][14] There have been several reports of modification of the L-chain 12,14,15) and the P25 protein, 13) but there have been no reports of modification of the H-chain in silkworms by this transgenic technique. Furthermore, these modifications of the L-chain and P25 have not demonstrated changes in the physical properties of fibroin fiber.…”

Preparation of recombinant proteins in milk to improve human and animal health

“…Interestingly, a recent work showed than human proteins can be secreted with silk. They are bound to one of the silk proteins and they can be released in water by a mild treatment [47].…”