Skip to main content
Log in

Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

In contrast to mammalian transglutaminases (TGs), plant members of the superfamily are poorly characterized. In order to produce pure and active TG for its functional and structural studies, variants of maize chloroplast transglutaminase (TGZ, Patent WWO03102128) were sub-cloned into a pET28 vector and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were present mainly as insoluble inclusion bodies. The TGZ4p variant with four B-type repeats (M r∼55 kDa), was affinity purified from urea-solubilized inclusion bodies. TGZ4p was refolded by rapid dilution in a Ca2+- and guanidine-containing buffer. Active TGZ4p shows the general catalytic characteristics described for other TGs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Armstrong N, De Lencastre A, Gouaux E (1999) A new protein folding screen: Application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. Protein Sci 8:1475–1483

    PubMed  CAS  Google Scholar 

  • Bernet E, Claparols I, Dondini L, Santos M, Serafini-Fracassini D, Torné JM (1999) Changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases (of initial differentiation) in maize calluses and their chloroplasts. Plant Physiol Biochem 37:899–909

    Article  CAS  Google Scholar 

  • Clarke LA, Rebelo CS, Gonçalves J, Boavida MG, Jordan P (2001) PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences. Mol Pathol 54:351–353

    Article  PubMed  CAS  Google Scholar 

  • Consoli L, Damerval C. (2001) Quantification of individual zein isoforms resolved by two-dimensional electrophoresis: Genetic variability in 45 maize inbred lines. Electrophoresis 22:2983–2989

    Article  PubMed  CAS  Google Scholar 

  • Cortez J, Bonner PL, Griffin M (2004) Application of transglutaminases in the modification of wool textiles. Enz Microb Tech 34:64–72

    Article  CAS  Google Scholar 

  • Della Mea M, Caparrós-Ruiz D, Claparols I, Serafini-Fracassini D, Rigau J (2004) AtPng1p. The first plant transglutaminase. Plant Physiol 135:2046–2054

    Article  PubMed  CAS  Google Scholar 

  • Hausch F, Halttunen T, Mäki M, Khosla C (2005) Design, synthesis and evaluation of gluten peptide analogs as selective inhibitors of human transglutaminase. Chem Biol 10:225–231

    Article  CAS  Google Scholar 

  • Icekson I, Apelbaum A. (1987) Evidence for transglutaminase activity in plant tissue. Plant Physiol 84:972–974

    Article  PubMed  CAS  Google Scholar 

  • Ichinose A, Souri M, Izumi T, Takahashi N (2000) Molecular and genetic mechanisms of factor XIII A subunit deficiency. Semin Thromb Hemost 26:5–10

    Article  PubMed  CAS  Google Scholar 

  • Ikura K, Kometani T, Sasaki R, Chiba H (1980) Crosslinking of soybean 7S and 11S proteins by transglutaminase. Agric Biol Chem 44:2979–2984

    CAS  Google Scholar 

  • Kim SW, Quinn-Allen MA, Camp JT, Macedo-Ribeiro S, Fuentes-Prior P, Bode W, Kane WH (2000). Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis. Biochemistry 39:1951–1958

    Article  PubMed  CAS  Google Scholar 

  • Kuraishi C, Yamazaki K, Susa Y (2001) Transglutaminase: its utilization in the food industry. Foods Rev Int 17:221–246

    Article  CAS  Google Scholar 

  • Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Nature Rev Mol Cell Biol 4:140–156

    Article  CAS  Google Scholar 

  • Pandey A, Andersen JS, Mann M (2000) Use of mass spectrometry to study signaling pathways. Sci STKE 2000: PL1

  • Torné JM, Santos M, Talavera D, Villalobos E (2002) Maize nucleotide sequence coding for a protein with transglutaminase activity and use thereof. ES Patent WO03102128 A1, 11 Dec 2003

  • Villalobos E, Torné JM, Rigau J, Ollés I, Claparols I, Santos M (2001) Immunogold localization of a transglutaminase related to grana development in different maize cell types. Protoplasma 216:155–163

    PubMed  CAS  Google Scholar 

  • Villalobos E, Santos M, Talavera D, Rodríguez-Falcón M, Torné JM (2004) Molecular cloning and characterization of a maize transglutaminase complementary DNA. Gene 336:93–104

    Article  PubMed  CAS  Google Scholar 

  • Yee VC, Pedersen LC, Le Trong I, Bishop PD, Stenkamp RE, Teller DC (1994) Three-dimensional structure of a transglutaminase: human blood coagulation factor XIII. Proc Natl Acad Sci USA 91:7296–7300

    Article  PubMed  CAS  Google Scholar 

  • Yokoyama K, Nio N, Kikuchi Y. (2004) Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol 64:447–454

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by an IQS-CEUB pre-doctoral grant to P.K. Carvajal-Vallejos and the Spanish project MCYT BFI2003-003318. A. Campos acknowledges a post-doctoral grant from Fundação Para a Ciência e Tecnologia of Ministério da Ciência, Tecnologia e Ensino Superior of Portugal (SFRH/BPD/20783/2004).We thank N. Cortadellas (Microscopy Services, UB); P. Casas and E. Prats (Antibodies Service, IBMB. CSIC); P. Marco (IIQAB, CSIC) for AB/18QA design; L. de Oliveira (Proteomic Services, PC, UB); A. Coehlo (ITQB, Universidade Nova, Lisbon, Portugal) for the revision of the manuscript; L. Valero (Peptide Synthesis Services, Univ. PF); M. Miquel and A. Sanchez (DNA Sequencing Services and Image Service, IBMB, CSIC) and Shirley Burgess for English corrections. We also thank CERBA (Generalitat de Catalunya) for partial financial support.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mireya Santos.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Carvajal-Vallejos, P.K., Campos, A., Fuentes-Prior, P. et al. Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli . Biotechnol Lett 29, 1255–1262 (2007). https://doi.org/10.1007/s10529-007-9377-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-007-9377-7

Keywords

Navigation