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

详细信息    查看全文

• 作者：Shaozhou Zhu (1)
  Cuiyu Gong (1)
  Lu Ren (1)
  Xingzhou Li (1) (2)
  Dawei Song (1)
  Guojun Zheng (1)
  
• 关键词：Inclusion bodies ; His ; tag ; Uridine phosphorylase ; (+) ; γ ; Lactamase ; (? ; γ ; Lactamase
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2013
• 出版时间：January 2013
• 年：2013
• 卷：97
• 期：2
• 页码：837-845
• 全文大小：414KB
• 参考文献：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):2654-659. doi:10.1073/pnas.042684199 CrossRef
  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 259-82
  3. Carrió MM, Villaverde A (2003) Role of molecular chaperones in inclusion body formation. FEBS Lett 537(1-):215-21 CrossRef
  4. Carrió MM, Cubarsi R, Villaverde A (2000) Fine architecture of bacterial inclusion bodies. FEBS Lett 471(1):7-1 CrossRef
  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):223-33 CrossRef
  6. C?té 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):2387-394. doi:10.1007/s00253-011-3562-2 CrossRef
  7. De Bernardez CE (1998) Refolding of recombinant proteins. Curr Opin Biotech 9(2):157-63 CrossRef
  8. de Groot NS, Ventura S (2006) Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Lett 580(27):6471-476 CrossRef
  9. de Groot NS, Espargaró A, More M, Ventura S (2008) Studies on bacterial inclusion bodies. Future Microbiol 3(4):423-35. doi:10.2217/17460913.3.4.423 CrossRef
  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 79:9-8 CrossRef
  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):3-3. doi:10.1002/bit.260410103 CrossRef
  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):65-0 CrossRef
  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):2408-418. doi:10.1111/j.1742-4658.2011.08163.x CrossRef
  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):134-42 CrossRef
  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):185-93 CrossRef
  16. Hannig G, Makrides SC (1998) Strategies for optimizing heterologous protein expression in / Escherichia coli. Trends Biotechnol 16(2):54-0 CrossRef
  17. Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381(6583):571-80 CrossRef
  18. Hochuli E, D?beli H, Schacher A (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr A 411:177-84 CrossRef
  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-):194-01 CrossRef
  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):1702-708
  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):83-01. doi:10.1093/dnares/6.2.83 CrossRef
  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):1065-072. doi:10.1007/s00253-006-0556-6 CrossRef
  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):614-22. doi:10.1007/s12010-008-8493-8
  24. Lilie H, Schwarz E, Rudolph R (1998) Advances in refolding of proteins produced in E. coli. Curr Opin. Biotech 9(5):497-01
  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):1185-189. doi:10.1126/science.1092612 CrossRef
  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):184-92 CrossRef
  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):584-91 CrossRef
  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):75-3. doi:10.1007/s12033-008-9136-6 CrossRef
  29. ?ali A, Potterton L, Yuan F, van Vlijmen H, Karplus M (1995) Evaluation of comparative protein modeling by MODELLER. Proteins 23(3):318-26. doi:10.1002/prot.340230306 CrossRef
  30. Schügerl K, Hubbuch J (2005) Integrated bioprocesses. Curr Opin Microbiol 8(3):294-00 CrossRef
  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):166-72 CrossRef
  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):75-2
  33. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99(4):303-10 CrossRef
  34. S?rensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in / Escherichia coli. J Biotechnol 115(2):113-28 CrossRef
  35. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biot 60(5):523-33. doi:10.1007/s00253-002-1158-6
  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):211-22. doi:10.1007/s00253-006-0465-8 CrossRef
  37. Tsumoto K, Ejima D, Kumagai I, Arakawa T (2003) Practical considerations in refolding proteins from inclusion bodies. Protein Expres Purif 28(1):1- CrossRef
  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):721-29. doi:10.1007/s00253-008-1716-7 CrossRef
  
• 作者单位：Shaozhou Zhu (1)
  Cuiyu Gong (1)
  Lu Ren (1)
  Xingzhou Li (1) (2)
  Dawei Song (1)
  Guojun Zheng (1)
  
  1. State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing, 100029, People’s Republic of China
  2. Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, People’s Republic of China
  
• ISSN：1432-0614

文摘

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.