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Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin

Paper ID Volume ID Publish Year Pages File Format Full-Text
25273 43564 2007 14 PDF Available
Title
Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin
Abstract

In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0 μg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8 mg of 99%-pure rHSA from 2 g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.

Keywords
HSA, human serum albumin; rHSA, recombinant HSA; PSG, posterior silk gland; MSG, middle silk gland; BmNPV, Bombyx mori nuclear polyhedrosis virus; CD, circular dichroism; nHSA, native HSA; AA, amino acid; PAGE, polyacrylamide gel electrophoresis; PBS, pho
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Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: Production of recombinant human serum albumin
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 128, Issue 3, 20 February 2007, Pages 531–544
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us