Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells

Milada Horynová, Kazuo Takahashi, Stacy Hall, Matthew B. Renfrow, Jan Novak, Milan Raška

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50 mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5 mg of GalNAc-T2 protein per 1 L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8 kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.

Original languageEnglish
Pages (from-to)175-180
Number of pages6
JournalProtein Expression and Purification
Volume81
Issue number2
DOIs
Publication statusPublished - 01-02-2012

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Galactosamine
Transferases
Insects
Peptides
Immunoglobulins
Uridine Diphosphate
Baculoviridae
Culture Media
Proteins
Immunoglobulin kappa-Chains
Sf9 Cells
Tromethamine
HEK293 Cells
Enzymes
Recombinant Proteins
Polysaccharides
Chromatography
Amino Acid Sequence
Cell Culture Techniques

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Horynová, Milada ; Takahashi, Kazuo ; Hall, Stacy ; Renfrow, Matthew B. ; Novak, Jan ; Raška, Milan. / Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells. In: Protein Expression and Purification. 2012 ; Vol. 81, No. 2. pp. 175-180.
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Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells. / Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B.; Novak, Jan; Raška, Milan.

In: Protein Expression and Purification, Vol. 81, No. 2, 01.02.2012, p. 175-180.

Research output: Contribution to journalArticle

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