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

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Abstract

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.

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Acknowledgments

We thank Dr. T. Yoshimura, Graduate School of Bioagricultural Sciences, Nagoya University for the frozen sections. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

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Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan

    Akifumi Mizutani, Hiroyuki Tsunashima, Ken-ichi Nishijima, Takako Sasamoto, Yuki Yamada, Yasuhiro Kojima, Makoto Motono, Jun Kojima, Yujin Inayoshi, Katsuhide Miyake & Shinji Iijima

  2. Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Enoch Y. Park

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  1. Akifumi Mizutani
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  2. Hiroyuki Tsunashima
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Correspondence to Ken-ichi Nishijima.

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11248_2011_9511_MOESM1_ESM.pdf

Supplemental Fig. 1 (a) Intracellular localization of chicken GalTs was analyzed by immunostaining. FLAG-tagged chicken GalTs were transiently expressed in Hela cells. GalTs were stained using anti-FLAG M2 antibody and TRITC-conjugated secondary antibody (red), and nuclei were counterstained with DAPI (blue). (b) Confocal microscopic immunofluorescence images of Hela cells that expressed GalT1-FLAG or GalT1-KDEL-FLAG. Endoplasmic reticulum retention signal KDEL sequence was attached at the C-terminal of GalT1 by PCR. After transfection of expression plasmids, cells were immunostained using anti-FLAG M2 antibody and FITC-conjugated secondary antibody. Z-stack images are shown (PDF 172 kb)

11248_2011_9511_MOESM2_ESM.pdf

Supplemental Fig. 2 (a, b) Average copy numbers of the integrated viral vector in blood cell (a) and magnum tissue (b) were measured by quantitative PCR for eGFP. *, not detected; WT, wild-type chicken. (c) Relative GalT1 mRNA amounts in the magnum of GM chickens. GAPDH mRNA was used for normalization and the results are represented as relative values against the amount of GalT1 mRNA in wild-type (WT) chicken. (d) Protein G-purified scFvFcs produced by GalT1-GM chickens were treated with or without β-galactosidase, then, subjected to RCA120 lectin blotting. scFvFc from transgenic chicken expressing scFvFc alone (Kamihira et al. 2005) was used as a control. Anti-Fc antibody was used for detection of the total amount of scFvFcs (PDF 53 kb)

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Mizutani, A., Tsunashima, H., Nishijima, Ki. et al. Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white. Transgenic Res 21, 63–75 (2012). https://doi.org/10.1007/s11248-011-9511-0

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