Skip to main content
SpringerLink
Log in

Novel soluble expression technologies derived from unique properties of halophilic proteins

  • Mini-Review
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Recombinant production of mammalian cytoplasmic proteins plays a major role in developing pharmaceutical products. Here we describe two expression technologies using unique nature of halophilic bacteria. One of such properties of halophilic bacteria is accumulation of compatible solutes in the cytoplasm. As the compatible solutes enhance protein solubility and folding, one might utilize these bacteria for cytoplasmic soluble expression of recombinant proteins, as described in this review. Another uniqueness is high reversibility of thermally unfolded halophilic proteins. Here we show that one such protein, β-lactamase (BLA), is highly soluble both in the native and thermally unfolded states and reversibly refolds after thermal melting. This makes BLA as a potential fusion partner for soluble expression of target proteins. The BLA fusion technology is also introduced in the review.

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

Access this article

Log in via an institution

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

Notes

  1. The observed CD change due to unfolding or refolding reflects both its kinetics and the temperature change. As described in the figure legend, the temperature change occurs over the time period of 120 s and hence is too slow to measure the kinetics but too fast to measure equilibrium reaction.

References

  • Afendra AS, Vargas C, Nieto JJ, Drainas C (2004) Gene transfer and expression of recombinant proteins in moderately halophilic bacteria. Methods Mol Biol 267:209–223

    CAS  Google Scholar 

  • Arakawa T, Timasheff SN (1985) The stabilization of proteins by osmolytes. Biophys J 47:411–414

    Article  CAS  Google Scholar 

  • Arvanitis N, Vergas C, Tegos G, Perysinakis A, Nieto JJ, Ventosa A, Drainas C (1995) Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae. Appl Environ Microbiol 61:3821–3825

    CAS  Google Scholar 

  • Chatterjee DK, Esposito D (2006) Enhanced soluble protein expression using two new fusion tags. Protein Expr Purif 46:122–129

    Article  CAS  Google Scholar 

  • Cline SW, Doolittle WF (1992) Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons. J Bacteriol 174:1076–1080

    CAS  Google Scholar 

  • Douka E, Christogianni A, Koukkou AI, Afendra AS, Drainas C (2001) Use of a green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata. FEMS Microbiol Lett 201:221–227

    Article  CAS  Google Scholar 

  • Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4:32

    Article  Google Scholar 

  • Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358

    Article  CAS  Google Scholar 

  • Finn RD, Kapelioukh I, Paine MJ (2005) Rainbow tags: a visual tag systems for recombinant protein expression and purification. Biotechniques 38:387–392

    Article  CAS  Google Scholar 

  • Frillingos S, Linden A, Niehaus F, Vargas C, Nieto JJ, Ventosa A (2000) Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata. J Appl Microbiol 88:495–503

    Article  CAS  Google Scholar 

  • Galinski EA (1995) Osmoadaptation in bacteria. Adv Microb Physiol 37:272–328

    CAS  Google Scholar 

  • Holmes ML, Dyall-Smith ML (1990) A plasmid vector with a selectable marker for halophilic archaebacteria. J Bacteriol 173:756–761

    Google Scholar 

  • Holmes ML, Nuttall SD, Dyall-Smith ML (1991) Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J Bacteriol 173:3807–3813

    CAS  Google Scholar 

  • Ignatova Z, Gierasch LM (2007) Effects of osmolytes on protein folding and aggregation in cells. Methods Enzymol 428:355–372

    Article  CAS  Google Scholar 

  • Ishibashi M, Tokunaga H, Hiratsuka K, Yonezawa Y, Tsurumaru H, Arakawa T, Tokunaga M (2001) NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt. FEBS Lett 493:134–138

    Article  CAS  Google Scholar 

  • Ishibashi M, Sakashita K, Tokunaga H, Arakawa T, Tokunaga M (2003) Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte, trimethylamine N-oxide. J Protein Chem 22:345–351

    Article  CAS  Google Scholar 

  • Lam WL, Doolittle WF (1992) Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci USA 86:5478–5482

    Article  Google Scholar 

  • Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189

    Article  CAS  Google Scholar 

  • Minton AP (2005) Influence of macromolecular crowding upon the stability and state of association of proteins: prediction and observation. J Pharm Sci 94:1668–1675

    Article  CAS  Google Scholar 

  • Mothet JP, Parent AT, Wolosker H, Brady RO Jr, Linden DJ, Ferris CD, Rogawski MA, Snyder SH (2000) d-serine is an endogenous ligand for the glycine site of the N-methyl-d-aspartate receptor. Proc Natl Acad Sci USA 97:4926–4931

    Article  CAS  Google Scholar 

  • Nagayoshi C, Ishibashi M, Kita Y, Matsuoka M, Nishimoto I, Tokunaga M (2005) Expression, refolding and characterization of human brain serine racemase in Escherichia coli with N-terminal His-tag. Protein Pept Lett 12:487–490

    Article  CAS  Google Scholar 

  • Nagayoshi C, Tokunaga H, Hayashi A, Harazono H, Hamasaki K, Ando A, Tokunaga M (2006) Efficient expression of haloarchaeal nucleoside diphosphate kinase via strong porin promoter in moderately halophilic bacteria. Protein Pept Lett 13:611–615

    Article  CAS  Google Scholar 

  • Nagayoshi C, Ishibashi M, Tokunaga M (2009) Purification and characterization of human brain serine racemase expressed in moderately halophilic bacteria. Protein Pept Lett 16:201–206

    Article  CAS  Google Scholar 

  • Ni BF, Chang M, Duschl A, Lanyi J, Needleman R (1990) An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium. Gene 90:169–172

    Article  CAS  Google Scholar 

  • Nomura S, Harada Y (1998) Functional expression of green fluorescent protein derivatives in Halobacterium salinarum. FEMS Microbiol Lett 167:287–293

    Article  CAS  Google Scholar 

  • Oren A, Larimer F, Richardson P, Lapidus A, Csonka LN (2005) How to be moderately halophilic with broad salt tolerance: clues from the genome of Chromohalobacter salexigens. Extremophiles 9:275–279

    Article  CAS  Google Scholar 

  • Patenge N, Haase A, Bolhuis H, Oesterhelt D (2000) The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum. Mol Microbiol 36:105–113

    Article  CAS  Google Scholar 

  • Qu Y, Bolen CL, Bolen DW (1998) Osmolyte-driven contraction of a random coil protein. Proc Natl Acad Sci USA 95:9628–9273

    Article  Google Scholar 

  • Kennedy SP, Ng WP, Salzberg SL, Hood L, DasSarma S (2001) Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence. Genome Res 11:1641–1650

    Article  CAS  Google Scholar 

  • Söhlemann P, Soppa J, Oesterhelt D, Lohse MJ (1997) Expression of beta 2-adrenoceptors in halobacteria. Naunyn-Schmiedebergs Arch Pharmacol 355:150–160

    Article  Google Scholar 

  • Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533

    CAS  Google Scholar 

  • Tokunaga H, Ishibashi M, Arakawa T, Tokunaga M (2004) Highly efficient renaturation of beta-lactamase isolated from moderately halophilic bacteria. FEBS Lett 558:7–12

    Article  CAS  Google Scholar 

  • Tokunaga H, Arakawa T, Fukada H, Tokunaga M (2006) Opposing effects of NaCl on reversibility and thermal stability of halophilic beta-lactamase from a moderate halophile, Chromohalobacter sp. 560. Biophys Chem 119:316–320

    Article  CAS  Google Scholar 

  • Tokunaga H, Arakawa T, Tokunaga M (2008) Engineering of halophilic enzymes: two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases. Protein Sci 17:1603–1610

    Article  CAS  Google Scholar 

  • Tokunaga H, Saito S, Sakai K, Yamaguchi R, Katsuyama I, Arakawa T, Onozaki K, Arakawa T, Tokunaga M (2010) Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin. Appl Microbiol Biotechnol 86:649–658

    Article  CAS  Google Scholar 

  • Vargas C, Nieto JJ (2004) Genetic tools for the manipulation of moderately halophilic bacteria of the family Halomonadaceae. Methods Mol Biol 267:183–208

    CAS  Google Scholar 

  • Vargas C, Fernández-Castillo R, Cánovas D (1995) Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. elongata and its use for shuttle vector construction. Mol Gen Genet 246:411–418

    Article  CAS  Google Scholar 

  • Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62:504–544

    CAS  Google Scholar 

  • Waugh DS (2005) Making the most of affinity tags. Treds Biotechnol 23:316–320

    Article  CAS  Google Scholar 

  • Widmann M, Christen P (2000) Comparison of folding rates of homologous prokaryotic and eukaryotic proteins. J Biol Chem 275:18619–18622

    Article  CAS  Google Scholar 

  • Wilkinson DL, Harrison RG (1991) Predicting the solubility of recombinant proteins in Escherichia coli. Bio/Technology 9:443–448

    Article  CAS  Google Scholar 

  • Wolosker H (2007) NMDA receptor regulation by d-serine: new findings and perspectives. Mol Neurobiol 36:152–164

    Article  CAS  Google Scholar 

  • Yonezawa Y, Izutsu K, Tokunaga H, Maeda H, Arakawa T, Tokunaga M (2007) Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetrameric Pseudomonas counterpart. FEMS Microbiol Lett 268:52–58

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan

    Hiroko Tokunaga & Masao Tokunaga

  2. Alliance Protein Laboratory, 3957 Corte Cancion, Thousand Oaks, CA, 91360, USA

    Tsutomu Arakawa

Authors
  1. Hiroko Tokunaga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tsutomu Arakawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masao Tokunaga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masao Tokunaga.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tokunaga, H., Arakawa, T. & Tokunaga, M. Novel soluble expression technologies derived from unique properties of halophilic proteins. Appl Microbiol Biotechnol 88, 1223–1231 (2010). https://doi.org/10.1007/s00253-010-2832-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2832-8

Keyword

Search

Navigation