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Zea mays L. transglutaminase expression in Escherichia coli

Background

Transglutaminase (protein-glutamine:amine γ-glutamyl-transferase, E.C. 2.3.2.13) catalyses acyl-transfer reactions between γ-carboxyamide groups of glutamine residues and the ε -amino group of lysines in proteins, leading to inter- or intramolecular cross-linking. Transglutaminases (TGs) have been found in mammals, plants, fish, nematodes and bacteria. Two maize cDNA clones (TGZ15 and TGZ21) that expressed active transglutaminase localized in chloroplasts were isolated [1, 2].

Results

A TGZ sequence was subcloned into pET (Novagen) vector (named as pET28a+TGZ4). The expression assays in E. coli BL21 DE3 cells showed that the main fraction of the protein (>80%) was found in the inclusion bodies (see Figure 1). The purification under denaturing conditions in FPLC system followed by a refolding step was a suitable procedure to obtain functional TGZ4p (see Figures 2,3). In addition, a specific antibody against TGZ4p was obtained in the laboratory for the immunolocalization of this protein in E. coli cells (see Figure 4).

Figure 1
figure1

Western blot of TGZ4p. S = soluble, IB = inslusion bodies

Figure 2
figure2

TGZ4p FPLC purification under denaturing conditions.

Figure 3
figure3

TGase activity of refolded TGZ4p against Tris and phosphate buffer.

Figure 4
figure4

TGZ4p TEM Immunolocalization, S=soluble, IB=inclusion body.

Conclusion

TGZ4p was expressed in E. coli mainly as inclusion bodies. The purification under denaturing conditions and refolding in vitro was a suitable procedure to obtain functional TG.

References

  1. 1.

    Villalobos E, Santos M, Talavera D, Rodríguez-Falcón M, Torné J: Molecular cloning and characterization of a maize transglutaminase complementary DNA. Gene. 2004, 336: 93-104. 10.1016/j.gene.2004.03.025.

  2. 2.

    Torné JM, Santos MA, Talavera D, Villalobos E: Maize nucleotide sequense coding for a protein with transglutaminase activity and use thereof. PCT/ES03/00247. Patent number WO03102128. 2002

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Acknowledgements

Thanks to the Spanish National Project MCYT BFI2003-003318 and CEUB-IQS fellowship.

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Correspondence to Patricia Carvajal.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Carvajal, P., Villalobos, E., Campos, A. et al. Zea mays L. transglutaminase expression in Escherichia coli. Microb Cell Fact 5, P8 (2006). https://doi.org/10.1186/1475-2859-5-S1-P8

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Keywords

  • Maize
  • Lysine
  • Glutamine
  • cDNA Clone
  • Inclusion Body