Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.
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 50mM 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 (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa 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.
Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.
Milada
M
Horynová
Kazuo
K
Takahashi
Stacy
S
Hall
Matthew B
MB
Renfrow
Jan
J
Novak
Milan
M
Raška
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media / Q000378:metabolism; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007070:Immunoglobulin A / Q000235:genetics / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry* / Q000235:genetics; D007313:Insecta / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D053719:Tandem Mass Spectrometry; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D003470:Culture Media; D007070:Immunoglobulin A; D007145:Immunoglobulin kappa-Chains; D017350:N-Acetylgalactosaminyltransferases; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase