Influence of signal-peptide truncations on the functional expression of Escherichia coli γ -glutamyltranspeptidase

✍ Scribed by Huei-Fen Lo; Wei-Mou Chou; Pei-Jing Chen; Long-Liu Lin

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: English
• Weight: 413 KB
• Volume: 48
• Category: Article
• ISSN: 0233-111X
• DOI: 10.1002/jobm.200700325

✦ Synopsis

Abstract

The full‐length Escherichia coli γ ‐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‐β ‐D‐thiogalactopyranoside induction of E. coli M15 cells bearing the recombinant plasmids resulted in the intracellular production of the expressed proteins, EcGGT, EcGGT/ΔN11, EcGGT/ΔN17, EcGGT/ΔN18, EcGGT/ΔN20, and EcGGT/ΔN26. The overexpressed enzymes were purified to near homogeneity by Ni^2+^‐NTA resin. The specific activity for EcGGT, EcGGT/ΔN11 and EcGGT/ΔN17 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/ΔN11 or pQE‐EcGGT/ΔN17. Western blot analysis confirmed that the majority of N‐terminally truncated enzymes were present in the cytoplasm. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)