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Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2012 Feb; Vol. 81 (2), pp. 175-80. Date of Electronic Publication: 2011 Oct 19. - Publication Year :
- 2012
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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 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.<br /> (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Animals
Baculoviridae genetics
Baculoviridae metabolism
Cloning, Molecular
Culture Media metabolism
Enzyme Activation
Genetic Vectors genetics
Genetic Vectors metabolism
HEK293 Cells
Humans
Immunoglobulin A genetics
Immunoglobulin A metabolism
Immunoglobulin kappa-Chains genetics
Insecta genetics
Insecta metabolism
Mice
Molecular Sequence Data
N-Acetylgalactosaminyltransferases genetics
N-Acetylgalactosaminyltransferases isolation & purification
Plasmids genetics
Plasmids metabolism
Protein Sorting Signals
Protein Stability
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins isolation & purification
Solubility
Tandem Mass Spectrometry
Transfection
Polypeptide N-acetylgalactosaminyltransferase
Immunoglobulin kappa-Chains chemistry
N-Acetylgalactosaminyltransferases biosynthesis
Recombinant Fusion Proteins biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0279
- Volume :
- 81
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 22033505
- Full Text :
- https://doi.org/10.1016/j.pep.2011.10.006