Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Expression of soluble recombinant transglutaminase from Zea mays in Pichia pastoris

  • 700 Accesses

  • 15 Citations

Abstract

Transglutaminases (TGases) catalyze post-translational protein modifications by ε-(γ-glutamyl) links and covalent amide bonds. In plant, this enzyme is poorly studied and only the Zea mays TGase gene (tgz) has been cloned. The tgz had been expressed in Escherichia coli, but the recombinant protein was mainly present in inclusion bodies. Therefore, to obtain active, soluble protein, we optimized its coding sequence according to the codon bias of Pichia pastoris and synthesized the sequence with SOEing-PCR. The optimized fragment was successfully transformed into P. pastoris GS115 by electroporation. The optimal conditions for expression were under a final concentration of 0.5 % methanol and a time-course of 96 h. The synthesized recombinant Zea mays transglutaminase (TGZs) was purified by affinity method, its production was 4.4 mg/L, and the specific activity was 0.889 U/mg under optimal expression condition. Optimal activity for TGZs was observed at 37 °C and a pH of 8.0, respectively. The cross-linking reaction of TGZs to the casein was studied, and the result was same as the reaction of casein by microbial transglutaminase. These results indicated that an effective procedure for expressing and purifying TGZs in P. pastoris GS115 was established.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

TGase:

Transglutaminase

tgz :

Zea mays transglutaminase gene

TGZs:

The synthesized recombinant Zea mays transglutaminase

MTG:

Microbial transglutaminase

TGZ:

Maize chloroplast transglutaminase

tgzs :

The synthesized Zea mays transglutaminase gene

References

  1. Ando H, Adachi M, Umeda K, Matsuura A, Nonaka M, Uchio R, Tanaka H, Motoki M (1989) Purification and characteristics of a novel transglutaminase derived from microorganisms. Agric Biol Chem 53(10):2613–2617

  2. Bellevik S, Summerer S, Meijer J (2002) Overexpression of Arabidopsis thaliana soluble epoxide hydrolase 1 in Pichia pastoris and characterisation of the recombinant enzyme. Protein Expr Purif 26(1):65–70

  3. Beninati S, Bergamini CM, Piacentini M (2009) An overview of the first 50 years of transglutaminase research. Amino Acids 36(4):591–598

  4. Carvajal P, Villalobos E, Campos A, Torné JM, Barberà E, Santos M (2006) Zea mays L. transglutaminase expression in Escherichia coli. Microb Cell Fact 5(Suppl 1):P8

  5. Carvajal P, Gibert J, Campos N, Lopera O, Barberà E, Torné JM, Santos M (2011) Activity of maize transglutaminase overexpressed in Escherichia coli inclusion bodies: an alternative to protein refolding. Biotechnol Prog 27(1):232–240

  6. Carvajal-Vallejos PK, Campos A, Fuentes-Prior P, Villalobos E, Almeida AM, Barbera E, Torné JM, Santos M (2007) Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli. Biotechnol Lett 29(8):1255–1262

  7. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24(1):45–66

  8. Chung S (1972) Comparative studies on tissue transglutaminase and factor XIII. Ann NY Acad Sci 202(1):240–255

  9. Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16(1):23–52

  10. Daly R, Hearn MTW (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18(2):119–138

  11. DeJong G, Koppelman S (2002) Transglutaminase catalyzed reactions: impact on food applications. J Food Sci 67(8):2798–2806

  12. Della Mea M, Caparrós-Ruiz D, Claparols I, Serafini-Fracassini D, Rigau J (2004) AtPng1p. The first plant transglutaminase. Plant Physiol 135(4):2046–2054

  13. Du G, Cui L, Zhu Y, Chen J (2007) Improvement of shrink-resistance and tensile strength of wool fabric treated with a novel microbial transglutaminase from Streptomyces hygroscopicus. Enzyme Microb Technol 40(7):1753–1757

  14. Dube M, Schäfer C, Neidhart S, Carle R (2007) Texturisation and modification of vegetable proteins for food applications using microbial transglutaminase. Eur Food Res Technol 225(2):287–299

  15. Folk J, Cole P (1966) Mechanism of action of guinea pig liver transglutaminase. J Biol Chem 241(23):5518

  16. Grossowicz N, Wainfan E, Borek E, Waelsch H (1950) The enzymatic formation of hydroxamic acids from glutamine and asparagine. J Biol Chem 187(1):111–125

  17. Hermanrud CE, Lucas CL, Sykes M, Huang CA, Wang Z (2011) Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris. Protein Expr Purif 76(1):115–120

  18. Ho ML, Leu SZ, Hsieh JF, Jiang ST (2000) Technical approach to simplify the purification method and characterization of microbial transglutaminase produced from Streptoverticillium ladakanum. J Food Sci 65(1):76–80

  19. Hu S, Li L, Qiao J, Guo Y, Cheng L, Liu J (2006) Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expr Purif 47(1):249–257

  20. Icekson I, Apelbaum A (1987) Evidence for transglutaminase activity in plant tissue. Plant Physiol 84(4):972

  21. Jin X, Meng N, Xia L (2011) Expression of an endo-β-1, 4-glucanase gene from Orpinomyces PC-2 in Pichia pastoris. Int J Mol Sci 12(5):3366–3380

  22. Lauber S, Henle T, Klostermeyer H (2000) Relationship between the crosslinking of caseins by transglutaminase and the gel strength of yoghurt. Eur Food Res Technol 210(5):305–309

  23. Lee SG, Koh HY, Han SJ, Park H, Na DC, Kim IC, Lee HK, Yim JH (2010) Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization. Protein Expr Purif 71(1):108–114

  24. Li J, Hong Y, Xiao Y, Xu Y, Fang W (2007) High production of laccase B from Trametes sp. in Pichia pastoris. World J Microb Biot 23(5):741–745

  25. Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 4(2):140–156

  26. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4):249–270

  27. Marx CK, Hertel TC, Pietzsch M (2008) Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme. Enzyme Microb Technol 42(7):568–575

  28. Masuda T, Tamaki S, Kaneko R, Wada R, Fujita Y, Mehta A, Kitabatake N (2004) Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris. Biotechnol Bioeng 85(7):761–769

  29. Moon JH, Hong YH, Huppertz T, Fox PF, Kelly AL (2009) Properties of casein micelles cross-linked by transglutaminase. Int J Dairy Technol 62(1):27–32

  30. Smiddy M, Martin JEGH, Kelly A, De Kruif C, Huppertz T (2006) Stability of casein micelles cross-linked by transglutaminase. J Dairy Sci 89(6):1906–1914

  31. Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190(1):55–62

  32. Torné Cubiró J, Santos Lozano M, Talavera Baro D, Villalobos Amador E, Rigau Lloveras J (2003) Maize nucleotide sequence coding for a protein with transglutaminase activity and use thereof. WO Patent WO/2003/102,128

  33. Villalobos E, Torné J, 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(3):155–163

  34. Villalobos E, Santos M, Talavera D, Rodrıguez-Falcón M, Torné J (2004) Molecular cloning and characterization of a maize transglutaminase complementary DNA. Gene 336(1):93–104

  35. Wu YW, Tsai YH (2006) A rapid transglutaminase assay for high-throughput screening applications. J Biomol Screen 11(7):836–843. doi:10.1177/1087057106291585

  36. Yang J, Liu L (2010) Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris. J Mol Catal B Enzym 63(3–4):164–169

  37. Yokoyama K, Nio N, Kikuchi Y (2004) Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol 64(4):447–454

  38. Yu Y-J, Wu S-C, Chan H–H, Chen Y-C, Chen Z-Y, Yang M-T (2008) Overproduction of soluble recombinant transglutaminase from Streptomyces netropsis in Escherichia coli. Appl Microbiol Biotechnol 81(3):523–532

  39. Zhang W, Yueqing C, Yuxian X (2008) Cloning of the subtilisin Pr1A gene from a strain of locust specific fungus, Metarhizium anisopliae, and functional expression of the protein in Pichia pastoris. World J Microb Biot 24(11):2481–2488

  40. Zhu Y, Tramper J (2008) Novel applications for microbial transglutaminase beyond food processing. Trends Biotechnol 26(10):559–565

Download references

Acknowledgments

This work was supported by National Nature Science Foundation of China (Grant No. 30972041).

Author information

Correspondence to Lanwei Zhang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 72 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, H., Zhang, L., Cui, Y. et al. Expression of soluble recombinant transglutaminase from Zea mays in Pichia pastoris . World J Microbiol Biotechnol 29, 939–947 (2013). https://doi.org/10.1007/s11274-012-1250-8

Download citation

Keywords

  • Transglutaminase
  • Zea mays
  • Codon optimization
  • Pichia pastoris GS115