Influence of N-terminal truncations on the functional expression of Bacillus licheniformis gamma-glutamyltranspeptidase in recombinant Escherichia coli.
The full-length Bacillus licheniformis gamma-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 vector pQE-30. Isopropyl-beta-D-thiogalactopyranoside induction of Escherichia coli M15 cells bearing the recombinant plasmids resulted in the overexpression of His(6)-tagged proteins BlGGT, BlGGT/DeltaN12, BlGGT/DeltaN43, BlGGT/DeltaN44, BlGGT/DeltaN45, BlGGT/DeltaN48, and BlGGT/DeltaN58. Except for BlGGT/DeltaN58, the overexpressed enzymes could be purified to near-homogeneity by Ni(2+)-NTA resin. The molecular masses of the precursor and subunits of BlGGT, BlGGT/DeltaN12, and BlGGT/DeltaN43 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/DeltaN12, BlGGT/DeltaN43, and BlGGT/DeltaN44 was 51.9+/-5.6, 1.3+/-0.2, 0.8+/-0.05, and 0.2+/-0.03 U/mg protein, respectively, whereas the remaining two enzymes had shown no GGT activity under the enzyme assay conditions. BlGGT, BlGGT/DeltaN12, BlGGT/DeltaN43, and BlGGT/DeltaN44 could process autocatalytically their precursors into alpha- and beta-subunits at 4 degrees C. These results indicate that removal of the signal peptide significantly affects the functional expression of BlGGT in recombinant E. coli.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan. llin@mail.ncyu.edu.tw
Yang
Li-Yu
LY
Hu
Hui-Yu
HY
Lo
Huei-Fen
HF
D001407:Bacillus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017384:Sequence Deletion; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase