Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

• Published in: Transgenic research 2012-02, Vol.21 (1), p.63-75
• Main Authors: Mizutani, Akifumi, Tsunashima, Hiroyuki, Nishijima, Ken-ichi, Sasamoto, Takako, Yamada, Yuki, Kojima, Yasuhiro, Motono, Makoto, Kojima, Jun, Inayoshi, Yujin, Miyake, Katsuhide, Park, Enoch Y, Iijima, Shinji
• Format: Article
• Language: English
• Subjects: Albumen; Amino Acid Sequence; Animal Genetics and Genomics; Animals; Animals, Genetically Modified; Antibodies; Base Sequence; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; biopharmaceuticals; Biotechnology; Breeding; chickens; Chickens - genetics; egg albumen; Egg Proteins - genetics; Egg Proteins - metabolism; Egg White; eggs; Erythropoietin; Female; Fundamental and applied biological sciences. Psychology; Galactose; Galactosyltransferases - genetics; Galactosyltransferases - metabolism; Gene Expression Regulation, Enzymologic; Gene transfer; Genetic Engineering; Genetic technics; Glycosylation; Golgi apparatus; Golgi Apparatus - genetics; Golgi Apparatus - metabolism; Gut-associated lymphoid tissues; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - metabolism; Life Sciences; Methods. Procedures. Technologies; Molecular Medicine; Molecular Sequence Data; N-linked glycans; necrosis; Original Paper; Oviducts - physiology; Pharmaceuticals; Plant Genetics and Genomics; polysaccharides; Protein Engineering - methods; protein synthesis; recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sialic acids; Single-Chain Antibodies - genetics; therapeutics; Tissue Distribution; Transgenic animals and transgenic plants; Transgenics; Tumor necrosis factor receptors
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-011-9511-0

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower β1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.