Abstract
Despite the importance of recombinant protein production in academy and industrial fields, many issues concerning the expression of soluble and homogeneous product are still unsolved. Although several strategies were developed to overcome these obstacles, at present there is no magic bullet that can be applied for all cases. Indeed, several key expression parameters need to be evaluated for each protein. Among the different hosts for protein expression, Escherichia coli is by far the most widely used. In this chapter, we review many of the different tools employed to circumvent protein insolubility problems.
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References
Sorensen HP (2010) Towards universal systems for recombinant gene expression. Microb Cell Fact 9:27
Huang CJ, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 39:383–399
Yang Z, Zhang L, Zhang Y et al (2011) Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PLoS One 6:e22981
Correa A, Oppezzo P (2011) Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J 6:715–730
Samuelson JC (2011) Recent developments in difficult protein expression: a guide to E. coli strains, promoters, and relevant host mutations. Methods Mol Biol 705:195–209
Vincentelli R, Cimino A, Geerlof A et al (2011) High-throughput protein expression screening and purification in Escherichia coli. Methods 55:65–72
Vincentelli R, Canaan S, Campanacci V et al (2004) High-throughput automated refolding screening of inclusion bodies. Protein Sci 13:2782–2792
Foit L, Morgan GJ, Kern MJ et al (2009) Optimizing protein stability in vivo. Mol Cell 36:861–871
Hart DJ, Waldo GS (2013) Library methods for structural biology of challenging proteins and their complexes. Curr Opin Struct Biol 23:403–408
Artimo P, Jonnalagedda M, Arnold K et al (2012) ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40:W597–W603
Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353
Puigbo P, Guzman E, Romeu A et al (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 35:W126–W131
Villalobos A, Ness JE, Gustafsson C et al (2006) Gene Designer: a synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics 7:285
Chung BK, Lee DY (2012) Computational codon optimization of synthetic gene for protein expression. BMC Syst Biol 6:134
Burgess-Brown NA, Sharma S, Sobott F et al (2008) Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study. Protein Expr Purif 59:94–102
Tegel H, Tourle S, Ottosson J et al (2010) Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3). Protein Expr Purif 69:159–167
Rosano GL, Ceccarelli EA (2009) Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microb Cell Fact 8:41
Marin M (2008) Folding at the rhythm of the rare codon beat. Biotechnol J 3:1047–1057
Voges D, Watzele M, Nemetz C et al (2004) Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system. Biochem Biophys Res Commun 318:601–614
Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946–950
Salis HM (2011) The ribosome binding site calculator. Methods Enzymol 498:19–42
Makino T, Skretas G, Georgiou G (2011) Strain engineering for improved expression of recombinant proteins in bacteria. Microb Cell Fact 10:32
Salinas G, Pellizza L, Margenat M et al (2011) Tuned Escherichia coli as a host for the expression of disulfide-rich proteins. Biotechnol J 6:686–699
Ferre F, Clote P (2005) DiANNA: a web server for disulfide connectivity prediction. Nucleic Acids Res 33:W230–W232
Lin HH, Tseng LY (2010) DBCP: a web server for disulfide bonding connectivity pattern prediction without the prior knowledge of the bonding state of cysteines. Nucleic Acids Res 38:W503–W507
Berkmen M (2012) Production of disulfide-bonded proteins in Escherichia coli. Protein Expr Purif 82:240–251
Klint JK, Senff S, Saez NJ et al (2013) Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli. PLoS One 8:e63865
Mergulhao FJ, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23:177–202
den Blaauwen T, Driessen AJ (1996) Sec-dependent preprotein translocation in bacteria. Arch Microbiol 165:1–8
Luirink J, Sinning I (2004) SRP-mediated protein targeting: structure and function revisited. Biochim Biophys Acta 1694:17–35
Natale P, Bruser T, Driessen AJ (2008) Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—distinct translocases and mechanisms. Biochim Biophys Acta 1778:1735–1756
Wagner S, Klepsch MM, Schlegel S et al (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105:14371–14376
Schlegel S, Rujas E, Ytterberg AJ et al (2013) Optimizing heterologous protein production in the periplasm of E coli by regulating gene expression levels. Microb Cell Fact 12:24
de Marco A (2009) Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb Cell Fact 8:26
Bessette PH, Aslund F, Beckwith J et al (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci U S A 96:13703–13708
Lobstein J, Emrich CA, Jeans C et al (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 11:56
Hatahet F, Nguyen VD, Salo KE et al (2010) Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microb Cell Fact 9:67
Nguyen VD, Hatahet F, Salo KE et al (2010) Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E. coli. Microb Cell Fact 10:1
Nozach H, Fruchart-Gaillard C, Fenaille F et al (2013) High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli. Microb Cell Fact 12:37
Walls D, Loughran ST (2011) Tagging recombinant proteins to enhance solubility and aid purification. Methods Mol Biol 681:151–175
Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7:620–634
Murphy MB, Doyle SA (2005) High-throughput purification of hexahistidine-tagged proteins expressed in E. coli. Methods Mol Biol 310:123–130
Zhu XQ, Li SX, He HJ et al (2005) On-column refolding of an insoluble His6-tagged recombinant EC-SOD overexpressed in Escherichia coli. Acta Biochim Biophys Sin (Shanghai) 37:265–269
Li M, Su ZG, Janson JC (2004) In vitro protein refolding by chromatographic procedures. Protein Expr Purif 33:1–10
Schafer F, Romer U, Emmerlich M et al (2002) Automated high-throughput purification of 6xHis-tagged proteins. J Biomol Tech 13:131–142
Vincentelli R, Canaan S, Offant J et al (2005) Automated expression and solubility screening of His-tagged proteins in 96-well format. Anal Biochem 346:77–84
Steen J, Uhlen M, Hober S et al (2006) High-throughput protein purification using an automated set-up for high-yield affinity chromatography. Protein Expr Purif 46:173–178
Magnusdottir A, Johansson I, Dahlgren LG et al (2009) Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates. Nat Methods 6:477–478
Bolanos-Garcia VM, Davies OR (2006) Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Biochim Biophys Acta 1760:1304–1313
Robichon C, Luo J, Causey TB et al (2011) Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography. Appl Environ Microbiol 77:4634–4646
Andersen KR, Leksa NC, Schwartz TU (2013) Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins 81:1857–1861
Schmidt TG, Skerra A (2007) The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2:1528–1535
Lichty JJ, Malecki JL, Agnew HD et al (2005) Comparison of affinity tags for protein purification. Protein Expr Purif 41:98–105
Schmidt TG, Batz L, Bonet L et al (2013) Development of the Twin-Strep-tag(R) and its application for purification of recombinant proteins from cell culture supernatants. Protein Expr Purif 92:54–61
Hammarstrom M, Hellgren N, van Den Berg S et al (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci 11:313–321
Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358
Pattenden LK, Thomas WG (2008) Amylose affinity chromatography of maltose-binding protein: purification by both native and novel matrix-assisted dialysis refolding methods. Methods Mol Biol 421:169–189
Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40
Dyson MR, Shadbolt SP, Vincent KJ et al (2004) Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnol 4:32
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 8:1668–1674
Cho HJ, Lee Y, Chang RS et al (2008) Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli. Protein Expr Purif 60:37–45
LaVallie ER, Lu Z, Diblasio-Smith EA et al (2000) Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. Methods Enzymol 326:322–340
Kim S, Lee SB (2008) Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners. Protein Expr Purif 62:116–119
LaVallie ER, DiBlasio EA, Kovacic S et al (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N Y) 11:187–193
Marblestone JG, Edavettal SC, Lim Y et al (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189
Malakhov MP, Mattern MR, Malakhova OA et al (2004) SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics 5:75–86
Butt TR, Edavettal SC, Hall JP et al (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43:1–9
Zhang Z, Li ZH, Wang F et al (2002) Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli. Protein Expr Purif 26:218–228
De Marco V, Stier G, Blandin S et al (2004) The solubility and stability of recombinant proteins are increased by their fusion to NusA. Biochem Biophys Res Commun 322:766–771
Nallamsetty S, Waugh DS (2006) Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr Purif 45:175–182
van den Berg S, Lofdahl PA, Hard T et al (2006) Improved solubility of TEV protease by directed evolution. J Biotechnol 121:291–298
Kapust RB, Tozser J, Copeland TD et al (2002) The P1′ specificity of tobacco etch virus protease. Biochem Biophys Res Commun 294:949–955
Moon AF, Mueller GA, Zhong X et al (2010) A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction. Protein Sci 19:901–913
Suzuki N, Hiraki M, Yamada Y et al (2010) Crystallization of small proteins assisted by green fluorescent protein. Acta Crystallogr D Biol Crystallogr 66:1059–1066
Smyth DR, Mrozkiewicz MK, McGrath WJ et al (2003) Crystal structures of fusion proteins with large-affinity tags. Protein Sci 12:1313–1322
Corsini L, Hothorn M, Scheffzek K et al (2008) Thioredoxin as a fusion tag for carrier-driven crystallization. Protein Sci 17:2070–2079
Esposito D, Garvey LA, Chakiath CS (2009) Gateway cloning for protein expression. Methods Mol Biol 498:31–54
Berrow NS, Alderton D, Sainsbury S et al (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35:e45
Unger T, Jacobovitch Y, Dantes A et al (2010) Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172:34–44
Correa A, Ortega C, Obal G, Alzari P, Vincentelli R, Oppezzo P (2014) Generation of a vector suite for protein solubility screening. Front Microbiol. 5: 67
Erijman A, Dantes A, Bernheim R et al (2011) Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J Struct Biol 175:171–177
Bond SR, Naus CC (2012) RF-Cloning.org: an online tool for the design of restriction-free cloning projects. Nucleic Acids Res 40:W209–W213
Vera A, Gonzalez-Montalban N, Aris A et al (2007) The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng 96:1101–1106
Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234
Blommel PG, Becker KJ, Duvnjak P et al (2007) Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition. Biotechnol Prog 23:585–598
Ukkonen K, Mayer S, Vasala A et al (2013) Use of slow glucose feeding as supporting carbon source in lactose autoinduction medium improves the robustness of protein expression at different aeration conditions. Protein Expr Purif 91:147–154
Krause M, Ukkonen K, Haataja T et al (2010) A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb Cell Fact 9:11
Vincentelli R, Romier C (2013) Expression in Escherichia coli: becoming faster and more complex. Curr Opin Struct Biol 23:326–334
Koehn J, Hunt I (2009) High-throughput protein production (HTPP): a review of enabling technologies to expedite protein production. Methods Mol Biol 498:1–18
Ventura S, Villaverde A (2006) Protein quality in bacterial inclusion bodies. Trends Biotechnol 24:179–185
Dechavanne V, Barrillat N, Borlat F et al (2010) A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions. Protein Expr Purif 75:192–203
Clark EDB (1998) Refolding of recombinant proteins. Curr Opin Biotechnol 9:157–163
Achmuller C, Kaar W, Ahrer K et al (2007) N(pro) fusion technology to produce proteins with authentic N termini in E. coli. Nat Methods 4:1037–1043
Ke T, Liang S, Huang J et al (2012) A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes. BMC Biotechnol 12:10
Tokatlidis K, Dhurjati P, Millet J et al (1991) High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D. FEBS Lett 282:205–208
Garcia-Fruitos E, Gonzalez-Montalban N, Morell M et al (2005) Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Fact 4:27
de Groot NS, Ventura S (2006) Protein activity in bacterial inclusion bodies correlates with predicted aggregation rates. J Biotechnol 125:110–113
Peternel S, Grdadolnik J, Gaberc-Porekar V et al (2008) Engineering inclusion bodies for non denaturing extraction of functional proteins. Microb Cell Fact 7:34
Garcia-Fruitos E (2010) Inclusion bodies: a new concept. Microb Cell Fact 9:80
Garcia-Fruitos E, Vazquez E, Diez-Gil C et al (2012) Bacterial inclusion bodies: making gold from waste. Trends Biotechnol 30:65–70
Villaverde A, Garcia-Fruitos E, Rinas U et al (2012) Packaging protein drugs as bacterial inclusion bodies for therapeutic applications. Microb Cell Fact 11:76
Low C, Moberg P, Quistgaard EM et al (2013) High-throughput analytical gel filtration screening of integral membrane proteins for structural studies. Biochim Biophys Acta 1830:3497–3508
Sala E, de Marco A (2010) Screening optimized protein purification protocols by coupling small-scale expression and mini-size exclusion chromatography. Protein Expr Purif 74:231–235
Hattori M, Hibbs RE, Gouaux E (2012) A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20:1293–1299
Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14:673–681
Backmark AE, Olivier N, Snijder A et al (2013) Fluorescent probe for high-throughput screening of membrane protein expression. Protein Sci 22:1124–1132
Dale GE, Broger C, Langen H et al (1994) Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase. Protein Eng 7:933–939
Eijsink VG, Bjork A, Gaseidnes S et al (2004) Rational engineering of enzyme stability. J Biotechnol 113:105–120
Rasila TS, Pajunen MI, Savilahti H (2009) Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment. Anal Biochem 388:71–80
Stemmer WP (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389–391
Roodveldt C, Aharoni A, Tawfik DS (2005) Directed evolution of proteins for heterologous expression and stability. Curr Opin Struct Biol 15:50–56
Waldo GS, Standish BM, Berendzen J et al (1999) Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17:691–695
Pedelacq JD, Piltch E, Liong EC et al (2002) Engineering soluble proteins for structural genomics. Nat Biotechnol 20:927–932
Cabantous S, Terwilliger TC, Waldo GS (2005) Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol 23:102–107
Pedelacq JD, Cabantous S, Tran T et al (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88
Maxwell KL, Mittermaier AK, Forman-Kay JD et al (1999) A simple in vivo assay for increased protein solubility. Protein Sci 8:1908–1911
Sieber V, Martinez CA, Arnold FH (2001) Libraries of hybrid proteins from distantly related sequences. Nat Biotechnol 19:456–460
Dahlroth SL, Nordlund P, Cornvik T (2006) Colony filtration blotting for screening soluble expression in Escherichia coli. Nat Protoc 1:253–258
Cornvik T, Dahlroth SL, Magnusdottir A et al (2005) Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli. Nat Methods 2:507–509
Acknowledgments
This work was financed by a research grant from FCE-7273 and FMV-7323, 2011 from Agencia Nacional de Investigación e Innovación (ANII), Montevideo, Uruguay to P. Oppezzo. A. Correa was financed by a doctoral fellowship from ANII, Uruguay.
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Correa, A., Oppezzo, P. (2015). Overcoming the Solubility Problem in E. coli: Available Approaches for Recombinant Protein Production. In: García-Fruitós, E. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 1258. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2205-5_2
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