Applied Microbiology and Biotechnology

, Volume 97, Issue 2, pp 837–845 | Cite as

A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression

  • Shaozhou Zhu
  • Cuiyu Gong
  • Lu Ren
  • Xingzhou Li
  • Dawei Song
  • Guojun Zheng
Methods and protocols
First Online:
Received:
Revised:
Accepted:

Abstract

The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (−)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (−)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.

Keywords

Inclusion bodies His-tag Uridine phosphorylase (+)-γ-Lactamase (−)-γ-Lactamase 
This is a preview of subscription content, log in to check access.

References

  1. Braun P, Hu Y, Shen B, Halleck A, Koundinya M, Harlow E, LaBaer J (2002) Proteome-scale purification of human proteins from bacteria. P Natl Acad Sci 99(5):2654–2659. doi: 10.1073/pnas.042684199 CrossRefGoogle Scholar
  2. Burgess RR, Richard RB, Murray PD (2009) Refolding solubilized inclusion body proteins. In: Methods of enzymology. Academic, Salt Lake. Chapter 17, vol 463, pp 259–282Google Scholar
  3. Carrió MM, Villaverde A (2003) Role of molecular chaperones in inclusion body formation. FEBS Lett 537(1–3):215–221CrossRefGoogle Scholar
  4. Carrió MM, Cubarsi R, Villaverde A (2000) Fine architecture of bacterial inclusion bodies. FEBS Lett 471(1):7–11CrossRefGoogle Scholar
  5. Coler RN, Dillon DC, Skeiky YAW, Kahn M, Orme IM, Lobet Y, Reed SG, Alderson MR (2009) Identification of Mycobacterium tuberculosis vaccine candidates using human CD4+ T-cells expression cloning. Vaccine 27(2):223–233CrossRefGoogle Scholar
  6. Côté G, Skory C (2011) Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Appl Microbiol Biot 93(6):2387–2394. doi: 10.1007/s00253-011-3562-2 CrossRefGoogle Scholar
  7. De Bernardez CE (1998) Refolding of recombinant proteins. Curr Opin Biotech 9(2):157–163CrossRefGoogle Scholar
  8. de Groot NS, Ventura S (2006) Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Lett 580(27):6471–6476CrossRefGoogle Scholar
  9. de Groot NS, Espargaró A, More M, Ventura S (2008) Studies on bacterial inclusion bodies. Future Microbiol 3(4):423–435. doi: 10.2217/17460913.3.4.423 CrossRefGoogle Scholar
  10. Dolatabadian A, Sanavy SAMM, Ghanati F, Gresshoff PM (2012) Morphological and physiological response of soybean treated with the microsymbiont Bradyrhizobium japonicum pre-incubated with genistein. S Afr J Bot 79:9–18CrossRefGoogle Scholar
  11. Fischer B, Sumner I, Goodenough P (1993) Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnol Bioeng 41(1):3–13. doi: 10.1002/bit.260410103 CrossRefGoogle Scholar
  12. García-Fruitós E, Vázquez E, Díez-Gil C, Corchero JL, Seras-Franzoso J, Ratera I, Veciana J, Villaverde (2011) A bacterial inclusion bodies: making gold from waste. Trends Biotechnol 30(2):65–70CrossRefGoogle Scholar
  13. Gatti-Lafranconi P, Natalello A, Ami D, Doglia SM, Lotti M (2011) Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology. FEBS J 278(14):2408–2418. doi: 10.1111/j.1742-4658.2011.08163.x CrossRefGoogle Scholar
  14. Griswold KE, Mahmood NA, Iverson BL, Georgiou G (2003) Effects of codon usage versus putative 5-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm. Protein Expres Purif 27(1):134–142CrossRefGoogle Scholar
  15. Haacke A, Fendrich G, Ramage P, Geiser M (2009) Chaperone over-expression in Escherichia coli: apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates. Protein Expres Purif 64(2):185–193CrossRefGoogle Scholar
  16. Hannig G, Makrides SC (1998) Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol 16(2):54–60CrossRefGoogle Scholar
  17. Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381(6583):571–580CrossRefGoogle Scholar
  18. Hochuli E, Döbeli H, Schacher A (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr A 411:177–184CrossRefGoogle Scholar
  19. Horchani H, Ouertani S, Gargouri Y, Sayari A (2009) The N-terminal His-tag and the recombination process affect the biochemical properties of Staphylococcus aureus lipase produced in Escherichia coli. J Mol Catal B-Enzym 61(3–4):194–201CrossRefGoogle Scholar
  20. Judd AK, Schneider M, Sadowsky MJ, de Bruijn FJ (1993) Use of repetitive sequences and the polymerase chain reaction technique to classify genetically related Bradyrhizobium japonicum serocluster 123 strains. Appl Environ Microb 59(6):1702–1708Google Scholar
  21. Kawarabayasi Y, Hino Y, Horikawa H, Yamazaki S, Haikawa Y, Jin-no K, Takahashi M, Sekine M, S-i B, Ankai A, Kosugi H, Hosoyama A, Fukui S, Nagai Y, Nishijima K, Nakazawa H, Takamiya M, Masuda S, Funahashi T, Tanaka T, Kudoh Y, Yamazaki J, Kushida N, Oguchi A, K-i A, Kubota K, Nakamura Y, Nomura N, Sako Y, Kikuchi H (1999) Complete Genome Sequence of an Aerobic Hyper-thermophilic Crenarchaeon, Aeropyrum pernix K1. DNA Res 6(2):83–101. doi: 10.1093/dnares/6.2.83 CrossRefGoogle Scholar
  22. Kirschner A, Altenbuchner J, Bornscheuer U (2007) Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli. Appl Microbiol Biot 73(5):1065–1072. doi: 10.1007/s00253-006-0556-6 CrossRefGoogle Scholar
  23. Li Y, Yang G, Huang X, Ye B, Liu M, Lin Z, Li C, Cao Z-a (2009) Recombinant Glutamine Synthetase (GS) from C. glutamicum Existed as Both Hexamers & Dedocamers and C-terminal His-tag Enhanced Inclusion Bodies Formation in E. coli. Appl Microbiol Biot 159(3):614–622. doi: 10.1007/s12010-008-8493-8 Google Scholar
  24. Lilie H, Schwarz E, Rudolph R (1998) Advances in refolding of proteins produced in E. coli. Curr Opin. Biotech 9(5):497–501Google Scholar
  25. Masip L, Pan JL, Haldar S, Penner-Hahn JE, DeLisa MP, Georgiou G, Bardwell JCA, Collet J-F (2004) An engineered pathway for the formation of protein disulfide bonds. Science 303(5661):1185–1189. doi: 10.1126/science.1092612 CrossRefGoogle Scholar
  26. Ohtaki A, Murata K, Sato Y, Noguchi K, Miyatake H, Dohmae N, Yamada K, Yohda M, Odaka M (2010) Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition. BBA-Proteins Proteom 1804(1):184–192CrossRefGoogle Scholar
  27. Park SH, Casagrande F, Chu M, Maier K, Kiefer H, Opella SJ (2011) Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination. BBA-Biomembranes 1818(3):584–591CrossRefGoogle Scholar
  28. Quintana-Castro R, Díaz P, Valerio-Alfaro G, García H, Oliart-Ros R (2009) Gene cloning, expression, and characterization of the Geobacillus thermoleovorans CCR11 thermoalkaliphilic lipase. Mol Biotechnol 42(1):75–83. doi: 10.1007/s12033-008-9136-6 CrossRefGoogle Scholar
  29. Šali A, Potterton L, Yuan F, van Vlijmen H, Karplus M (1995) Evaluation of comparative protein modeling by MODELLER. Proteins 23(3):318–326. doi: 10.1002/prot.340230306 CrossRefGoogle Scholar
  30. Schügerl K, Hubbuch J (2005) Integrated bioprocesses. Curr Opin Microbiol 8(3):294–300CrossRefGoogle Scholar
  31. Shuo-shuo C, Xue-zheng L, Ji-hong S (2011) Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase Lip-948. Protein Expres Purif 77(2):166–172CrossRefGoogle Scholar
  32. Singh SM, Sharma A, Upadhyay AK, Singh A, Garg LC, Panda AK (2011) Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form. Protein Expres Purif 81(1):75–82Google Scholar
  33. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99(4):303–310CrossRefGoogle Scholar
  34. Sørensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115(2):113–128CrossRefGoogle Scholar
  35. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biot 60(5):523–533. doi: 10.1007/s00253-002-1158-6 Google Scholar
  36. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biot 72(2):211–222. doi: 10.1007/s00253-006-0465-8 CrossRefGoogle Scholar
  37. Tsumoto K, Ejima D, Kumagai I, Arakawa T (2003) Practical considerations in refolding proteins from inclusion bodies. Protein Expres Purif 28(1):1–8CrossRefGoogle Scholar
  38. Wardenga R, Hollmann F, Thum O, Bornscheuer U (2008) Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones. Appl Microbiol Biot 81(4):721–729. doi: 10.1007/s00253-008-1716-7 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Shaozhou Zhu
    • 1
  • Cuiyu Gong
    • 1
  • Lu Ren
    • 1
  • Xingzhou Li
    • 1
    • 2
  • Dawei Song
    • 1
  • Guojun Zheng
    • 1
  1. 1.State Key Laboratory of Chemical Resources EngineeringBeijing University of Chemical TechnologyBeijingPeople’s Republic of China
  2. 2.Beijing Institute of Pharmacology and ToxicologyBeijingPeople’s Republic of China

Personalised recommendations