Volume 5 Supplement 1

The 4th Recombinant Protein Production Meeting: a comparative view on host physiology

Open Access

Zea mays L. transglutaminase expression in Escherichia coli

  • Patricia Carvajal1,
  • Enrique Villalobos2,
  • Alexandre Campos2,
  • JMa Torné2,
  • Eduard Barberà1 and
  • Mireya Santos2
Microbial Cell Factories20065(Suppl 1):P8

https://doi.org/10.1186/1475-2859-5-S1-P8

Published: 10 October 2006

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

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

Figure 2

TGZ4p FPLC purification under denaturing conditions.

Figure 3

TGase activity of refolded TGZ4p against Tris and phosphate buffer.

Figure 4

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.

Declarations

Acknowledgements

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

Authors’ Affiliations

(1)
Department of Chemical Engineering, Biotechnology lab.-GQBB group, Institut Químic de Sarrià (IQS), Universitat Ramon Llull
(2)
Institut de Biología Molecular de Barcelona-Consejo Superior de Investigaciones Científicas (IBMB-CSIC)

References

  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.View ArticleGoogle Scholar
  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. 2002Google Scholar

Copyright

© Carvajal et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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