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

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Abstract

The full-length Bacillus licheniformis γ-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-β-d-thiogalactopyranoside induction of Escherichia coli M15 cells bearing the recombinant plasmids resulted in the overexpression of His6-tagged proteins BlGGT, BlGGT/ΔN12, BlGGT/ΔN43, BlGGT/ΔN44, BlGGT/ΔN45, BlGGT/ΔN48, and BlGGT/ΔN58. Except for BlGGT/ΔN58, the overexpressed enzymes could be purified to near-homogeneity by Ni2+-NTA resin. The molecular masses of the precursor and subunits of BlGGT, BlGGT/ΔN12, and BlGGT/ΔN43 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/ΔN12, BlGGT/ΔN43, and BlGGT/ΔN44 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/ΔN12, BlGGT/ΔN43, and BlGGT/ΔN44 could process autocatalytically their precursors into α- and β-subunits at 4°C. These results indicate that removal of the signal peptide significantly affects the functional expression of BlGGT in recombinant E. coli.

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Acknowledgment

This work was supported by the National Science Council of Taiwan (NSC 95-2313-B-415-012-MY3).

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Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan

    Long-Liu Lin & Li-Yu Yang

  2. Department of Food and Nutrition, Hungkuang University, Shalu, Taichung, 433-01, Taiwan

    Hui-Yu Hu & Huei-Fen Lo

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  1. Long-Liu Lin
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  2. Li-Yu Yang
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  3. Hui-Yu Hu
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Correspondence to Long-Liu Lin.

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Lin, LL., Yang, LY., Hu, HY. et al. Influence of N-Terminal Truncations on the Functional Expression of Bacillus licheniformis γ-Glutamyltranspeptidase in Recombinant Escherichia coli . Curr Microbiol 57, 603–608 (2008). https://doi.org/10.1007/s00284-008-9250-5

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