Expression and Characterization of β-1,4-Galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae

摘要

The lgtB genes that encodeβ-1,4-galactosyltransferase from Neisseria meningitidis ATCC 13102 and gonorrhoeae ATCC 31151 were isolated by a polymerase chain reaction using the pfu DNA polymerase. They were expressed under the control of lac and T7 promoters in Escherichia coli M15 and BL21 (DE3). Although the genes were efficiently expressed in E. coli M15 at 37℃ (33 kDa), most the β-1,4-galactosyltransferases that were produced were insoluble and proteolysed into enzymatically inactive polypeptides that lacked C-terminal residues (29.5 kDa and 28 kDa) during the purification steps. When the temperature of the cell growth was lowered to 25℃, however, the solubility of the β-1,4-galactosyltransferases increased substantially. A stable N-terminal his-tagged recombinant enzyme preparation could be achieved with E. coli BL21 (DE3) that expressed lgtB. Therefore, the cloned β-1,4-galactosyltransferases were expressed under the control of the T7 promoter in E. coli BL21 (DE3), mostly to the soluble form at 25℃. The proteins were easily purified to homogeneity by column chromatography using Ni-NTA resin, and were found to be active. The galactosyltransferases exhibited pH optimum at 6.5-7.0, and had an essential requirement for the Mn~(+2) ions for its action. The Mg~(+2) and Ca~(+2) ions showed about half of the galactosyltransferase activities with the Mn~(+2) ion. In the presence of the Fe~(+2) ion, partial activation was observed with the β-1,4-galactosyltransferase from N. meningitidis (64% of the enzyme activity with the Mn~(+2) ion), but not from N. gonorrhoeae. On the other hand, the Ni~(+2), Zn~(+2), and Cu~(+2) ions could not activate the β-1,4-galactosyltransferase activity. The inhibited enzyme activity with the Ni~(+2) ion was partially recovered with the Mn~(+2) ion, but in the presence of the Fe~(+2), Zn~(+2), and Cu~(+2) ions, the Mn~(+2) ion could not activate the enzyme activities. Also, the β-1,4-galactosyltransferase activity was 1.5-fold stimulate with the non-ionic detergent Triton X-100 (0.1-5%).