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Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag

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Abstract

In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material. As an alternative, proteinase K was investigated. This protease was successfully applied for the activation of purified pro-rMTG either as free or immobilized enzyme and the free enzyme was also applicable directly in the crude cell extract of E. coli. Thus, it enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations. The protocol has been successfully applied to both, wild-type transglutaminase of Streptomyces mobaraensis as well as to the highly active variant S2P. Proteinase K activates the pro-rMTG without unwanted degradation of the histidine-tag. It turned out to be very important to inhibit proteinase K activity, e.g., by PMSF, prior to protein separation by SDS–PAGE.

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Abbreviations

BDM:

Bio dry mass

DNA:

Deoxyribonucleic acid

E. coli :

Escherichia coli

HESylation:

Modification of proteins by hydroxy ethyl starch (HES)

MALDI-MS:

Matrix assisted laser desorption ionization mass spectrometry

His-tag:

Histidine-tag

IMAC:

Immobilized metal affinity chromatography

MTG:

Microbial transglutaminase of Streptomyces mobaraensis

PMSF:

Phenylmethylsulfonyl fluoride

pro-rMTG:

Inactive pro-enzyme of the recombinant microbial transglutaminase

rMTG:

Recombinant microbial his-tagged transglutaminase (variant of Streptomyces mobaraensis TG)

rMTG(S2P):

Recombinant microbial his-tagged transglutaminase (thermostable variant of Streptomyces mobaraensis TG)

SDS–PAGE:

Sodium dodecylsulfate polyacrylamide gel electrophoresis

TG:

Transglutaminase

References

  • 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

    Article  CAS  Google Scholar 

  • Beninati S, Piacentini M (2004) The transglutaminase family: an overview: mini review article. Amino Acids 26(4):367–372

    PubMed  CAS  Google Scholar 

  • Besheer A, Hertel TC, Kressler J, Mäder K, Pietzsch M (2009) Enzymatically catalyzed HESylation using microbial transglutaminase: proof of feasibility. J Pharm Sci 98:4420–4428

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Buettner K, Hertel TC, Pietzsch M (2011) Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis. Amino Acids. doi:10.1007/s00726-011-1015-y

  • Cardamone JM (2007) Enzyme-mediated crosslinking of wool. part I: transglutaminase. Text Res J 77(4):214–221

    Article  CAS  Google Scholar 

  • Chou S-Y (2009) Transglutaminase for crosslinking antigen to generate polyvalent antigens as vaccines against human pathogens and for antibody production. US Patent 7485438

  • Date M, Yokoyama K, Umezawa Y, Matsui H, Kikuchi Y (2003) Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum. Appl Environ Microbiol 69(5):3011–3014

    Article  PubMed  CAS  Google Scholar 

  • Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H (1974) Proteinase K from Tritirachium album Limber. Eur J Biochem 47(1):91–97

    Article  PubMed  CAS  Google Scholar 

  • Folk JE (1969) Mechanism of action of guinea pig liver transglutaminase. VI. Order of substrate addition. J Biol Chem 244(13):3707–3713

    PubMed  CAS  Google Scholar 

  • Gross-Bellard M, Oudet P, Chambon P (1973) Isolation of high-molecular-weight DNA from mammalian cells. Eur J biochem 36(1):32–38

    Article  PubMed  CAS  Google Scholar 

  • Itaya H, Kikuchi Y (2008) Secretion of Streptomyces mobaraensis pro-transglutaminase by Coryneform bacteria. Appl Microbiol Biotechnol 78(4):621–625

    Article  PubMed  CAS  Google Scholar 

  • Kamata Y, Ishikawa E, Motoki M (1992) Enzyme immobilization on ion exchangers by forming an enzyme coating with transglutaminase as a crosslinker. Biosci Biotechnol Biochem 56(8):1323–1324

    Article  PubMed  CAS  Google Scholar 

  • Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H (2003) Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-like protease from Streptomyces albogriseolus. Appl Environ Microbiol 69(1):358–366

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(259):680–685

    Article  PubMed  CAS  Google Scholar 

  • Mariniello L, Porta R (2005) Transglutaminases as biotechnological tools. Prog Exp Tumor Res 38:174–191

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Marx CK, Hertel TC, Pietzsch M (2008b) Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat sensitive variants. J Biotechnol 136:156–162

    Article  PubMed  CAS  Google Scholar 

  • Pasternack R, Dorsch S, Otterbach JT, Robenek IR, Wolf S, Fuchsbauer HL (1998) Bacterial pro-transglutaminase from Streptoverticillium mobaraense—purification, characterisation and sequence of the zymogen. Eur J Biochem 257(3):570–576

    Article  PubMed  CAS  Google Scholar 

  • Patzsch K, Riedel K, Pietzsch M (2010) Parameter optimization for protein film production using microbial transglutaminase. Biomacromolecules 11:896–903

    Article  PubMed  CAS  Google Scholar 

  • Portilla-Rivera OM, Tellez-Luis SJ, Ramirez de Leon JA, Vazquez M (2009) Production of microbial transglutaminase on media made from sugar cane molasses and glycerol. Food Technol Biotechnol 47(1):19–26

    CAS  Google Scholar 

  • Russell MW, Bergmeier LA, Zanders ED, Lehner T (1980) Protein antigens of Streptococcus mutans: purification and properties of a double antigen and its protease-resistant component. Infect Immun 28(2):486–493

    PubMed  CAS  Google Scholar 

  • Sato H (2002) Enzymatic procedure for site-specific pegylation of proteins. Adv Drug Deliv Rev 54(4):487–504

    Article  PubMed  CAS  Google Scholar 

  • Sommer C, Volk N, Pietzsch M (2011) Model based optimization of the fed-batch production of a highly active transglutaminase variant in E. coli. Protein Expr Purif 77:9–19

    Article  PubMed  CAS  Google Scholar 

  • Synowiecki J, Wolosowska S (2006) Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by crosslinking with transglutaminase. Enzyme Microb Technol 39(7):1417–1422

    Article  CAS  Google Scholar 

  • Yang H-L, Pan L, Lin Y (2009) Purification and on-column activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli. Biosci Biotechnol Biochem 73(11):2531–2534

    Article  PubMed  CAS  Google Scholar 

  • Zotzel J, Keller P, Fuchsbauer HL (2003) Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease. Eur J Biochem 270(15):3214–3222

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Pharmacy, Faculty of Sciences I, Martin Luther University Halle-Wittenberg, 06099, Halle (Saale), Germany

    Christian Sommer, Thomas C. Hertel, Christian E. H. Schmelzer & Markus Pietzsch

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  1. Christian Sommer
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  2. Thomas C. Hertel
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  3. Christian E. H. Schmelzer
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  4. Markus Pietzsch
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Correspondence to Markus Pietzsch.

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Sommer, C., Hertel, T.C., Schmelzer, C.E.H. et al. Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag. Amino Acids 42, 997–1006 (2012). https://doi.org/10.1007/s00726-011-1016-x

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  • DOI: https://doi.org/10.1007/s00726-011-1016-x

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