Functional expression of streptococcal galactosyltransferase in baculovirus/insect cell expression system.
The cpsIaJ gene of Streptococcus agalactiae type Ia codes for beta-1,4-galactosyltransferase. In this study, the functional expression of His-tagged CpsIaJ in a baculovirus expression system was performed, because the efficient functional expression of this enzyme in Escherichia coli had been unsuccessful. Using a partially purified enzyme preparation, we found that the enzyme had a restricted substrate specificity and that the entire structure of the substrate GlcNAc beta1-3Gal beta1-4Glc was required for the activity. Furthermore, mutations in a conserved DXD motif caused the loss of the enzyme's activity.
Kataoka
Yohei
Y
Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
Ozeki
Shingo
S
Miyake
Katsuhide
K
Iijima
Shinji
S
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000737:chemistry; D015870:Gene Expression; D007313:Insecta; D009154:Mutation; D013291:Streptococcus / Q000201:enzymology*; D013292:Streptococcus agalactiae / Q000378:metabolism; D013379:Substrate Specificity
D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase