Influence of signal-peptide truncations on the functional expression of Escherichia coli gamma -glutamyltranspeptidase.
The full-length Escherichia coli gamma -glutamyltranspeptidase (EcGGT) gene and five truncations lacking 33, 51, 54, 60, and 78 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 E. coli M15 cells bearing the recombinant plasmids resulted in the intracellular production of the expressed proteins, EcGGT, EcGGT/DeltaN11, EcGGT/DeltaN17, EcGGT/DeltaN18, EcGGT/DeltaN20, and EcGGT/DeltaN26. The overexpressed enzymes were purified to near homogeneity by Ni(2+)-NTA resin. The specific activity for EcGGT, EcGGT/DeltaN11 and EcGGT/DeltaN17 was 5.3, 4.9, and 4.8 U/mg protein respectively, whereas the rest three enzymes had shown no GGT activity under the enzyme assay conditions. More than 94% of the activity was found in the cytoplasmic fraction of E. coli M15 cells harboring pQE-EcGGT, pQE-EcGGT/DeltaN11 or pQE-EcGGT/DeltaN17. Western blot analysis confirmed that the majority of N-terminally truncated enzymes were present in the cytoplasm.
Lo
Huei-Fen
HF
Department of Food and Nutrition, Hungkuang University, Shalu, Taichung, Taiwan.
Chou
Wei-Mou
WM
Chen
Pei-Jing
PJ
Lin
Long-Liu
LL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase