Applied Microbiology and Biotechnology

, Volume 103, Issue 5, pp 2205–2216 | Cite as

The NT11, a novel fusion tag for enhancing protein expression in Escherichia coli

  • Thi Khoa My Nguyen
  • Mi Ran Ki
  • Ryeo Gang Son
  • Seung Pil PackEmail author
Biotechnologically relevant enzymes and proteins


The Escherichia coli (E. coli) expression system has been widely used to produce recombinant proteins. However, in some heterologous expressions, there are still difficulties in large-scale production. The use of fusion partners is one of the strategies for improving the expression levels of proteins in E. coli host. Here, we demonstrate a novel fusion element, the NT11-tag, which enhances protein expression. The NT11-tag was derived from the first 11 amino acid residues within the N-terminal N-half domain of a duplicated carbonic anhydrase (dCA) from Dunaliella species. Previously, we have found that the tag improves expression of the C-half domain of dCA when linked to its N-terminus. To verify its use as a protein production enhancer tag, two kinds of CAs derived from Hahella chejuensis (Hc-CA) and Thermovibrio ammonifican (Ta-CA) and the yellow fluorescent protein (YFP) were used as model proteins to measure their increased expression upon fusion with the NT11-tag. The NT11-tag amplified protein expression in E. coli by 6.9- and 7.6-fold for Ta-CA and YFP, respectively. Moreover, the tag also enhanced the soluble expression of Hc-CA, Ta-CA, and YFP by 1.7-, 5.0-, and 3.2-fold, respectively. Furthermore, protein yield was increased without inhibiting protein function. These results indicate that the use of the NT11-tag is a promising method for improving protein production in E. coli.


Fusion partner Solubility enhancement tag Esterase activity CO2 hydration 


Funding information

This work was supported by the Basic Core Technology Development Program for the Oceans and the Polar Regions of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning, Korea (NRF-2015M1A5A1037054) and a Marine Biomaterials Research Center grant from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Korea. This work was also supported by Research Fellow Funding grant funded by the Ministry of Education, Korea (NRF-2014R1A1A2008088) and BK21 plus.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2018_9595_MOESM1_ESM.pdf (643 kb)
ESM 1 (PDF 643 kb)


  1. Allen GS, Zavialov A, Gursky R, Ehrenberg M, Frank J (2005) The cryo-EM structure of a translation initiation complex from Escherichia coli. Cell 121(5):703–712. PubMedGoogle Scholar
  2. Bellaousov S, Reuter JS, Seetin MG, Mathews DH (2013) RNAstructure: web servers for RNA secondary structure prediction and analysis. Nucleic Acids Res 41(Web Server issue):W471–W474. PubMedPubMedCentralGoogle Scholar
  3. Bivona L, Zou ZC, Stutzman N, Sun PD (2010) Influence of the second amino acid on recombinant protein expression. Protein Expr Purif 74(2):248–256. PubMedPubMedCentralGoogle Scholar
  4. Butt TR, Edavettal SC, Hall JP, Mattern MR (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43(1):1–9. PubMedGoogle Scholar
  5. Cheng Y, Gu JA, Wang HG, Yu S, Liu YQ, Ning YL, Zou QM, Yu XJ, Mao XH (2010) EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli. J Biotechnol 150(3):380–388. PubMedGoogle Scholar
  6. Chou CP (2007) Engineering cell physiology to enhance recombinant protein production in Escherichia coli. Appl Microbiol Biotechnol 76(3):521–532. PubMedGoogle Scholar
  7. Christendat D, Yee A, Dharamsi A, Kluger Y, Savchenko A, Cort JR, Booth V, Mackereth CD, Saridakis V, Ekiel I, Kozlov G, Maxwell KL, Wu N, McIntosh LP, Gehring K, Kennedy MA, Davidson AR, Pai EF, Gerstein M, Edwards AM, Arrowsmith CH (2000) Structural proteomics of an archaeon. Nat Struct Biol 7(10):903–909. PubMedGoogle Scholar
  8. Costa SJ, Almeida A, Castro A, Domingues L, Besir H (2013) The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology. Appl Microbiol Biotechnol 97(15):6779–6791. PubMedGoogle Scholar
  9. Costa SJ, Almeida A, Castro A, Domingues L (2014) Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Front Microbiol 5:63. PubMedPubMedCentralGoogle Scholar
  10. Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27(3):297–306. PubMedGoogle Scholar
  11. Di Fiore A, Alterio V, Monti SM, De Simone G, D'Ambrosio K (2015) Thermostable carbonic anhydrases in biotechnological applications. Int J Mol Sci 16(7):15456–15480. PubMedPubMedCentralGoogle Scholar
  12. Douette P, Navet R, Gerkens P, Galleni M, Levy D, Sluse FE (2005) Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL. Biochem Bioph Res Co 333(3):686–693. Google Scholar
  13. 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. PubMedPubMedCentralGoogle Scholar
  14. Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17(4):353–358. PubMedGoogle Scholar
  15. Hu J, Qin H, Sharma M, Cross TA, Gao FP (2008) Chemical cleavage of fusion proteins for high-level production of transmembrane peptides and protein domains containing conserved methionines. Biochim Biophys Acta 1778(4):1060–1066. PubMedGoogle Scholar
  16. James P, Isupov MN, Sayer C, Saneei V, Berg S, Lioliou M, Kotlar HK, Littlechild JA (2014) The structure of a tetrameric alpha-carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability. Acta Crystallogr D Biol Crystallogr 70(Pt 10):2607–2618. PubMedGoogle Scholar
  17. Jo BH, Seo JH, Cha HJ (2014) Bacterial extremo-alpha-carbonic anhydrases from deep-sea hydrothermal vents as potential biocatalysts for CO2 sequestration. J Mol Catal B-Enzym 109:31–39. Google Scholar
  18. Jones MD, Fayerman JT (1987) Industrial applications of recombinant-DNA technology. J Chem Educ 64(4):337–339. Google Scholar
  19. Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8(8):1668–1674. PubMedPubMedCentralGoogle Scholar
  20. Kato A, Maki K, Ebina T, Kuwajima K, Soda K, Kuroda Y (2007) Mutational analysis of protein solubility enhancement using short peptide tags. Biopolymers 85(1):12–18. PubMedGoogle Scholar
  21. Ki MR, Min K, Kanth BK, Lee J, Pack SP (2013) Expression, reconstruction and characterization of codon-optimized carbonic anhydrase from Hahella chejuensis for CO2 sequestration application. Bioprocess Biosyst Eng 36(3):375–381. PubMedGoogle Scholar
  22. Ki MR, Nguyen TKM, Kim SH, Kwon I, Pack SP (2016) Chimeric protein of internally duplicated alpha-type carbonic anhydrase from Dunaliella species for improved expression and CO2 sequestration. Process Biochem 51(9):1222–1229. Google Scholar
  23. Kohl T, Schmidt C, Wiemann S, Poustka A, Korf U (2008) Automated production of recombinant human proteins as resource for proteome research. Proteome Sci 6:4. PubMedPubMedCentralGoogle Scholar
  24. Kohyama K, Matsumoto T, Imoto T (2010) Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer. J Biochem 147(3):426–431. Google Scholar
  25. Kozak M (2005) Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361:13–37. PubMedGoogle Scholar
  26. Kudla G, Murray AW, Tollervey D, Plotkin JB (2009) Coding-sequence determinants of gene expression in Escherichia coli. Science 324(5924):255–258. PubMedPubMedCentralGoogle Scholar
  27. Kudou M, Yunmioka R, Ejima D, Arakawa T, Tsumoto K (2011) A novel protein refolding system using lauroyl-L-glutamate as a solubilizing detergent and arginine as a folding assisting agent. Protein Expr Purif 75(1):46–54. PubMedGoogle Scholar
  28. Li YF (2011) Self-cleaving fusion tags for recombinant protein production. Biotechnol Lett 33(5):869–881. PubMedGoogle Scholar
  29. 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(1):182–189. PubMedPubMedCentralGoogle Scholar
  30. Min KH, Son RG, Ki MR, Choi YS, Pack SP (2016) High expression and biosilica encapsulation of alkaline-active carbonic anhydrase for CO2 sequestration system development. Chemosphere 143:128–134. PubMedGoogle Scholar
  31. Ohtake S, Kita Y, Arakawa T (2011) Interactions of formulation excipients with proteins in solution and in the dried state. Adv Drug Deliv Rev 63(13):1053–1073. PubMedGoogle Scholar
  32. Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51(1):1–10. PubMedGoogle Scholar
  33. Ramos R, Moreira S, Rodrigues A, Gama M, Domingues L (2013) Recombinant expression and purification of the antimicrobial peptide magainin-2. Biotechnol Prog 29(1):17–22. PubMedGoogle Scholar
  34. Reddy RC, Lilie H, Rudolph R, Lange C (2005) L-arginine increases the solubility of unfolded species of hen egg white lysozyme. Protein Sci 14(4):929–935. Google Scholar
  35. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5(172).
  36. Seo SW, Yang JS, Kim I, Yang J, Min BE, Kim S, Jung GY (2013) Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency. Metab Eng 15:67–74. PubMedGoogle Scholar
  37. Sharma L, Sharma A (2001) Influence of cyclodextrin ring substituents on folding-related aggregation of bovine carbonic anhydrase. Eur J Biochem 268(8):2456–2463. PubMedGoogle Scholar
  38. Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature 254(5495):34–38. PubMedGoogle Scholar
  39. Sorensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115(2):113–128. PubMedGoogle Scholar
  40. Su Y, Zou ZR, Feng SY, Zhou P, Cao LJ (2007) The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli. J Biotechnol 129(3):373–382. PubMedGoogle Scholar
  41. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60(5):523–533. PubMedGoogle Scholar
  42. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72(2):211–222. PubMedGoogle Scholar
  43. Tessema M, Simons PC, Cimino DF, Sanchez L, Waller A, Posner RG, Wandinger-Ness A, Prossnitz ER, Sklar LA (2006) Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH. Cytometry A 69(5):326–334. PubMedGoogle Scholar
  44. Timasheff SN (2002) Protein hydration, thermodynamic binding, and preferential hydration. Biochemistry 41(46):13473–13482. PubMedGoogle Scholar
  45. Tsumoto K, Ejima D, Kumagai I, Arakawa T (2003) Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif 28(1):1–8. PubMedGoogle Scholar
  46. Umetsu M, Tsumoto K, Hara M, Ashish K, Goda S, Adschiri T, Kumagai I (2003) How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system—spectroscopic evidence for highly efficient refolding of a single-chain FV fragment. J Biol Chem 278(11):8979–8987. PubMedGoogle Scholar
  47. Vandevenne M, Gaspard G, Belgsir el M, Ramnath M, Cenatiempo Y, Marechal D, Dumoulin M, Frere JM, Matagne A, Galleni M, Filee P (2011) Effects of monopropanediamino-beta-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD. Biochim Biophys Acta 1814(9):1146–1153. PubMedGoogle Scholar
  48. Verpoorte JA (1967) Esterase activities of human carbonic anhydrases B and C. J Biol Chem 242(18):4221–4229PubMedGoogle Scholar
  49. Waugh DS (2005) Making the most of affinity tags. Trends Biotechnol 23(6):316–320. PubMedGoogle Scholar
  50. Waugh DS (2011) An overview of enzymatic reagents for the removal of affinity tags. Protein Expr Purif 80(2):283–293. PubMedPubMedCentralGoogle Scholar
  51. Wilbur KM, Anderson NG (1948) Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem 176(1):147–154PubMedGoogle Scholar
  52. Yamaguchi H, Miyazaki M, Briones-Nagata MP, Maeda H (2010) Refolding of difficult-to-fold proteins by a gradual decrease of denaturant using microfluidic chips. J Biochem 147(6):895–903. PubMedGoogle Scholar
  53. Yee A, Pardee K, Christendat D, Savchenko A, Edwards AM, Arrowsmith CH (2003) Structural proteomics: toward high-throughput structural biology as a tool in functional genomics. Acc Chem Res 36(3):183–189. PubMedGoogle Scholar
  54. Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7(5):620–634. PubMedGoogle Scholar
  55. Zhang YB, Howitt J, McCorkle S, Lawrence P, Springer K, Freimuth P (2004) Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expr Purif 36(2):207–216. PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Thi Khoa My Nguyen
    • 1
  • Mi Ran Ki
    • 1
  • Ryeo Gang Son
    • 1
  • Seung Pil Pack
    • 1
    Email author
  1. 1.Department of Biotechnology and BioinformaticsKorea UniversitySejongSouth Korea

Personalised recommendations