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Published on: July 11, 2013
Functional expression of streptococcal galactosyltransferase in baculovirus/insect cell expression system
Yohei Kataoka1, Shingo Ozeki, Katsuhide Miyake
1Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
Journal of Bioscience and Bioengineering
|May 24, 2006
Summary
Streptococcus agalactiae type Ia beta-1,4-galactosyltransferase (CpsIaJ) was functionally expressed in a baculovirus system. The enzyme requires a specific substrate structure and a conserved DXD motif for activity.
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- Streptococcus agalactiae type Ia possesses the cpsIaJ gene encoding a beta-1,4-galactosyltransferase.
- Efficient functional expression of this enzyme in Escherichia coli has been challenging.
Purpose of the Study:
- To achieve functional expression of the His-tagged CpsIaJ enzyme from Streptococcus agalactiae type Ia.
- To characterize the substrate specificity and essential motifs of the CpsIaJ enzyme.
Main Methods:
- Functional expression of His-tagged CpsIaJ using a baculovirus expression system.
- Enzyme activity assays using partially purified enzyme preparations.
- Site-directed mutagenesis to investigate the role of conserved motifs.
Main Results:
- Successful functional expression of CpsIaJ was achieved in the baculovirus system.
- The enzyme demonstrated restricted substrate specificity, requiring the complete GlcNAc beta1-3Gal beta1-4Glc structure.
- Mutations within a conserved DXD motif abolished enzyme activity, indicating its critical role.
Conclusions:
- The baculovirus system is suitable for functional expression of Streptococcus agalactiae type Ia beta-1,4-galactosyltransferase.
- The CpsIaJ enzyme's activity is dependent on a specific substrate conformation and the integrity of the DXD motif.
