Influence of N-Terminal Truncations on the Functional Expression of Bacillus licheniformis d-Glutamyltranspeptidase in Recombinant Escherichia coli

摘要

The full-length Bacillus licheniformis d-glutamyltranspeptidase (BlGGT) gene and six truncations lacking 36, 129, 132, 135, 144, and 174 bp, respectively, at the 5' end were prepared by polymerase chain reaction and cloned into the expression vectorpQE-30. Isopropyl-o-d-thiogalactopyranoside induction of Escherichia coli M15 cells bearing the recombinant plasmids resulted in the overexpression of His-tagged proteins BlGGT, BlGGT/N12, BlGGT/N43, BlGGT/N44, BlGGT/N45, BlGGT/N48, and BlGGT/N58. Except for BlGGT/N58, the overexpressed enzymes could be purified to near-homogeneity by Nipo-NTA resin. The molecular masses of the precursor and subunits of BlGGT, BlGGT/N12, and BlGGT/N43 were determined to be 63, 41, and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but other recombinant enzymes exhibited predominantly as a precursor form. The specific activity for purified BlGGT, BlGGT/N12, BlGGT/N43, and BlGGT/N44 was 51.9 pl 5.6, 1.3 pl 0.2, 0.8 pl 0.05, and 0.2 pl 0.03 U/mg protein, respectively, whereas the remaining two enzymes had shown no GGT activity under the enzyme assay conditions. BlGGT, BlGGT/N12, BlGGT/N43, and BlGGT/N44 could process autocatalytically their precursors into l- and o-subunits at 4pC. These results indicate that removal of the signal peptide significantly affects the functional expression of BlGGT in recombinant E. coli.