Skip to main content

Learning about protein solubility from bacterial inclusion bodies


The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed.


The conformational quality of soluble recombinant proteins is an emerging matter of concern, especially when the obtained products are to be used for functional or interactomic analyses [1]. In the context of recombinant protein production, the general believing that soluble protein species are properly folded and fully functional in contrast to the misfolded and inactive protein versions trapped in insoluble inclusion bodies [2], cannot be longer supported by current research data. The dropping of independent references to inclusion bodies as entities formed by functional protein species with native secondary structure is progressively increasing, and the structural and functional diversity of the model proteins used in these studies [313] does leave little room to speculate about this fact as being an artefact or a peculiarity of a limited number of protein species. Recent reviews in this area have presented properly folded proteins as natural components of inclusion bodies [10, 14], indirectly compromising the paradigm of recombinant protein solubility as equivalent to protein conformational quality [15].

Indeed, the occurrence of functional proteins as important components of bacterial aggregates prompts to reconsider the conformational quality of protein species occurring in the soluble cell fraction of inclusion body-forming cells, that might be lower than expected. Several indirect observations are also in this line; (i) the functional quality of recombinant proteins in E. coli is affected in parallel by physical parameters such as temperature (high temperature impairs protein activity in both soluble and insoluble cell fractions) [16] and physiological conditions such as the availability of chaperones (a molar excess of DnaKJ inactivates both soluble and insoluble recombinant proteins) [17]; (ii) in vivo disintegration of inclusion bodies is strongly dependent on proteolytic degradation [1821] for which DnaK is required [20], indicating a tight surveillance of the quality control system over aggregated protein species; (iii) inclusion body-forming proteins can complete their folding process once embedded in these aggregates [22]; (iv) the soluble versions of recombinant proteins can occur as soluble aggregates [23, 24]; (v) the functional quality (measured for a model enzyme as its specific activity and fluorescent proteins by specific emission) of soluble protein versions can be lower than that of the inclusion body counterparts [3], and be eventually improved by reducing the growth temperature of recombinant cells from 37 to 16°C [16]. This indicates that at 37°C, an important fraction of soluble protein species are inactive, suggesting that they have not reached their native conformation. This has been very recently explored by sub-fractioning the soluble population of an inclusion body-forming recombinant GFP and their subsequent functional analysis. Indeed, there is a large functional diversity within the soluble protein population (accompanied by an extremely high abundance of soluble aggregates, either globular or fibrilar) [24], that prompts to observe the specific fluorescence of the soluble protein version as an average rather than a canonical value defined by a single type of molecular species.

In this scenario, recombinant proteins in producing cells can be seen as adopting "a continuum of forms" [23] expanding from soluble to insoluble cell fractions, and inclusion bodies as insoluble "clusters" of protein species [19]. Therefore, soluble versions of a given protein would not necessarily show better conformational quality than the aggregated counterparts, although the average biological activity (specific activity for enzymes or specific fluorescence for fluorescent proteins) is in general higher in the soluble cell fraction [3, 24]. Interestingly, the specific enzymatic activities (or fluorescence emission) of soluble and insoluble protein versions tend to adopt similar values under specific conditions such as in DnaK knockout mutants [25, 26]. Therefore, the soluble and insoluble "virtual" cell fractions in bacteria [14] are now regarded as more virtual than ever, as the main feature distinguishing soluble and inclusion body protein species might be the dispersed-clustered status rather than the biological activity.

From a practical point of view, these emerging concepts about protein aggregation in recombinant bacteria have remarkable implications. First, inclusion bodies formed by enzymes can be straightforward used as catalysers in industry-relevant enzymatic reactions skipping any previous in vitro refolding protocols [57]. Second, the quality of inclusion body proteins can be dramatically enhanced by producing them at suboptimal temperatures. This should not only permit the production of inclusion bodies with improved catalyzing properties but it also might favour the controlled in vitro release of functional proteins from these aggregates. In this regard, the recovery of functional proteins from inclusion bodies has been a largely used strategy when a desired protein species showed a high aggregation tendency. Such an approach implies separation of inclusion bodies, efficient protein unfolding under extreme denaturation conditions and further refolding through complex (and often unsuccessful) step strategies to be optimized for any particular protein species [27]. However, in the last years, an increasing piece of evidence points out that inclusion bodies with high content of native-like structure could be easily solubilised in non-denaturing conditions avoiding strong denaturation and refolding steps. A set of non related proteins, namely GFP [28], archaeon proteins, cytokines, immunoglobulin-folded proteins [29] and β-2-microglobulin [30], have been successfully extracted from inclusion bodies without the need of denaturing conditions, basically using as solubilising agents L-arginine and GdnHCl at non-denaturing concentrations [28, 29]. Also in this line, Menart and co-workers observed that functional proteins could be easily extracted from inclusion bodies using non denaturing mild detergents and polar solvents, provided that the cells would have been cultured under suboptimal temperatures [12]. Such inclusion bodies, being a straightforward source of soluble proteins, were named "non-classical" because of their unexpected high content of functional, extractable species. Although sufficient data has been now accumulated to infer that in general, inclusion bodies are non-classical by nature (regarding the unlink between solubility and activity) [15], this interesting approach would potentially permit to skip complex refolding procedures by engineering the quality of inclusion body proteins during the production process. In very recent papers, Peternel and co-workers reported not only the successful extraction of functional polypeptides from inclusion bodies but also the fact that, in some cases, the biological activity of these inclusion body-solubilised proteins was comparable or even higher that than found in the soluble fraction. For instance, human granulocyte-stimulating factor (hG-CSF), GFP and lymphotoxin α (LT-α) extracted from inclusion bodies represented around the 98%, 40% and 25%, respectively, of the total biological activity and fluorescence emission in the recombinant protein producing-cells [31, 32]. Again, the different structural and biological properties of the proteins for which this principle has been proved indicate that the extractability of functional proteins from inclusion bodies is not a particular issue, although its applicability at large scale needs to be further evaluated. On the other side, as an additional strategy, the specific activity of inclusion body proteins can be successfully enhanced by down-regulating the levels of recombinant gene expression [33, 34].

Finally, since early recombinant DNA times, when the formation of inclusion bodies was noticed as a general undesirable event [35], enhancing protein solubility has been compulsory pursued through diverse approaches. The need for soluble proteins for many research, industrial and pharmaceutical applications has pushed microbiologists, biochemists and chemical engineers to modify cell, protein and process conditions (using protease-deficient cells, chaperone co-production, removing hydrophobic regions, fusion of solubility tags, minimizing the growth rate or using weak gene expression induction conditions among others), in an attempt to favour the occurrence of the target protein in the soluble cell fraction [3641]. However, solubility is often observed as an academic parameter, namely the quotient (in %) between soluble and total protein and therefore with a questionable practical value. Interestingly, it is very rare to find in the literature measures of solubility simultaneous to determinations of protein yield or functional quality, when attempting a novel strategy to minimize inclusion body formation, such as for instance, the co-production of chaperones along with the recombinant protein species. In this regard, enhancing the levels of trigger factor and GroELS increases the solubility of a recombinant lysozyme that shows a specific activity lower than in absence of additional chaperones [42]. Other chaperone sets have been observed to promote solubility of target proteins [40, 43, 44] without a detailed analysis of protein quality and activity or by determining specific activity referring it to cell extracts or total (recombinant or not) protein [45, 46]. Also, there are clear indications that the solubility enhancement under such conditions might eventually be associated to an increase of soluble aggregates [47]. Interestingly, lower protein yields obtained during chaperone co-production result in higher enzymatic activity in cell extracts and enhanced solubility, as observed by cyclodextrin glycosyltransferase [48] and mouse endostatin and human lysozyme respectively [42].

Furthermore, fine analyses of solubility in combination with other more useful parameters such as yield of soluble polypeptide or the biological activity reveal intriguing physiological events. For instance, co-production of the DnaKJ chaperone pair along with a target recombinant protein indeed favours solubility but at expenses of protein quality and yield [20]. In fact, enhancing the intracellular levels of DnaK, alone or within distinct chaperone sets (a common strategy to increase solubility) [40], dramatically diminishes protein stability through the stimulation of Lon- and ClpP-dependent proteolysis of inclusion body polypeptides [20]. In this regard, both yield and quality of a model recombinant GFP and other unrelated proteins are largely enhanced in DnaK- mutants [20, 25, 26], in which the solubility percent value is, as expected, lower than in wilt type hosts. More intriguingly, plotting solubility percent data versus protein yield of functional quality renders extremely good but negative correlations, under different genetic backgrounds [20] or production conditions [49]. Preliminary data about non bacterial protein production systems from our group obtained by M. Martínez Alonso (not shown) indicate that such a negative correlation between yield (or quality) and solubility could be a general issue. Therefore, when designing a protein production process the most pertinent strategy should be chosen depending on what parameter (yield, quality or solubility) is the most relevant to the final use of the protein. Eventually, recombinant protein solubility could be merely dependent on the intracellular concentration of the recombinant protein itself, what would ultimately fit with the enhanced solubility observed at low growth rates, low temperatures and weak doses of gene expression inductor [36, 41].

There are still exciting issues regarding bacterial inclusion bodies that deserve full scientific attention, such as the solving of the inner molecular organization that allows the occurrence of proper folded species within a general amyloid-like aggregate pattern [50, 51]. Also, the sequence-dependent nature of protein aggregation [5254] is still poorly known from a mechanistic point of view. From the biotechnological side, it is widely accepted that production of aggregation-prone protein triggers cell responses to conformational stress [5558], irrespective of the host used as cell factory [59]. If such set of physiological responses cannot be efficiently controlled, enhancing protein solubility without renouncing to protein quality might be then a mirage. Surfing the complex network of cell activities that regulate protein aggregation (for instance, through rational metabolic engineering) could be a choice strategy to approach the production of soluble and high quality recombinant proteins. For such a more gentle use of cell factories, a deeper comprehension of the recombinant cell physiology and quality control system is urgently needed.


  1. 1.

    de Marco A: Minimal information: an urgent need to assess the functional reliability of recombinant proteins used in biological experiments. Microb Cell Fact. 2008, 7: 20- 10.1186/1475-2859-7-20.

    Article  Google Scholar 

  2. 2.

    Baneyx F, Mujacic M: Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol. 2004, 22: 1399-1408. 10.1038/nbt1029.

    CAS  Article  Google Scholar 

  3. 3.

    Garcia-Fruitos E, Gonzalez-Montalban N, Morell M, Vera A, Ferraz RM, Aris A, et al: Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Fact. 2005, 4: 27- 10.1186/1475-2859-4-27.

    Article  Google Scholar 

  4. 4.

    Arie JP, Miot M, Sassoon N, Betton JM: Formation of active inclusion bodies in the periplasm of Escherichia coli. Mol Microbiol. 2006, 62: 427-437. 10.1111/j.1365-2958.2006.05394.x.

    CAS  Article  Google Scholar 

  5. 5.

    Nahalka J, Vikartovska A, Hrabarova E: A crosslinked inclusion body process for sialic acid synthesis. J Biotechnol. 2008, 134: 146-153. 10.1016/j.jbiotec.2008.01.014.

    CAS  Article  Google Scholar 

  6. 6.

    Nahalka J: Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to alpha-D: -glucose-1-phosphate. J Ind Microbiol Biotechnol. 2008, 35: 219-223. 10.1007/s10295-007-0287-4.

    CAS  Article  Google Scholar 

  7. 7.

    Nahalka J, Dib I, Nidetzky B: Encapsulation of Trigonopsis variabilis D-amino acid oxidase and fast comparison of the operational stabilities of free and immobilized preparations of the enzyme. Biotechnol Bioeng. 2008, 99: 251-260. 10.1002/bit.21579.

    CAS  Article  Google Scholar 

  8. 8.

    Nahalka J, Nidetzky B: Fusion to a pull-down domain: a novel approach of producing Trigonopsis variabilisD-amino acid oxidase as insoluble enzyme aggregates. Biotechnol Bioeng. 2007, 97: 454-461. 10.1002/bit.21244.

    CAS  Article  Google Scholar 

  9. 9.

    Nahalka J, Gemeiner P, Bucko M, Wang PG: Bioenergy beads: a tool for regeneration of ATP/NTP in biocatalytic synthesis. Artif Cells Blood Substit Immobil Biotechnol. 2006, 34: 515-521. 10.1080/10731190600862886.

    CAS  Article  Google Scholar 

  10. 10.

    Doglia SM, Ami D, Natalello A, Gatti-Lafranconi P, Lotti M: Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies. Biotechnol J. 2008, 3: 193-201. 10.1002/biot.200700238.

    CAS  Article  Google Scholar 

  11. 11.

    Garcia-Fruitos E, Aris A, Villaverde A: Localization of functional polypeptides in bacterial inclusion bodies. Appl Environ Microbiol. 2007, 73: 289-294. 10.1128/AEM.01952-06.

    CAS  Article  Google Scholar 

  12. 12.

    Jevsevar S, Gaberc-Porekar V, Fonda I, Podobnik B, Grdadolnik J, Menart V: Production of nonclassical inclusion bodies from which correctly folded protein can be extracted. Biotechnol Prog. 2005, 21: 632-639. 10.1021/bp0497839.

    CAS  Article  Google Scholar 

  13. 13.

    Oberg K, Chrunyk BA, Wetzel R, Fink AL: Nativelike secondary structure in interleukin-1 beta inclusion bodies by attenuated total reflectance FTIR. Biochemistry. 1994, 33: 2628-2634. 10.1021/bi00175a035.

    CAS  Article  Google Scholar 

  14. 14.

    Ventura S, Villaverde A: Protein quality in bacterial inclusion bodies. Trends Biotechnol. 2006, 24: 179-185. 10.1016/j.tibtech.2006.02.007.

    CAS  Article  Google Scholar 

  15. 15.

    Gonzalez-Montalban N, Garcia-Fruitos E, Villaverde A: Recombinant protein solubility-does more mean better?. Nat Biotechnol. 2007, 25: 718-720. 10.1038/nbt0707-718.

    CAS  Article  Google Scholar 

  16. 16.

    Vera A, Gonzalez-Montalban N, Aris A, Villaverde A: The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng. 2007, 96: 1101-1106. 10.1002/bit.21218.

    CAS  Article  Google Scholar 

  17. 17.

    Martinez-Alonso M, Vera A, Villaverde A: Role of the chaperone DnaK in protein solubility and conformational quality in inclusion body-forming Escherichia coli cells. FEMS Microbiol Lett. 2007, 273: 187-195. 10.1111/j.1574-6968.2007.00788.x.

    CAS  Article  Google Scholar 

  18. 18.

    Vera A, Aris A, Carrio M, Gonzalez-Montalban N, Villaverde A: Lon and ClpP proteases participate in the physiological disintegration of bacterial inclusion bodies. J Biotechnol. 2005, 119: 163-171. 10.1016/j.jbiotec.2005.04.006.

    CAS  Article  Google Scholar 

  19. 19.

    Rinas U, Hoffmann F, Betiku E, Estape D, Marten S: Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli. J Biotechnol. 2007, 127: 244-257. 10.1016/j.jbiotec.2006.07.004.

    CAS  Article  Google Scholar 

  20. 20.

    Garcia-Fruitos E, Martinez-Alonso M, Gonzalez-Montalban N, Valli M, Mattanovich D, Villaverde A: Divergent Genetic Control of Protein Solubility and Conformational Quality in Escherichia coli. J Mol Biol. 2007, 374: 195-205. 10.1016/j.jmb.2007.09.004.

    CAS  Article  Google Scholar 

  21. 21.

    Alcala P, Feliu JX, Aris A, Villaverde A: Efficient accommodation of recombinant, foot-and-mouth disease virus RGD peptides to cell-surface integrins. Biochem Biophys Res Commun. 2001, 285: 201-206. 10.1006/bbrc.2001.5157.

    CAS  Article  Google Scholar 

  22. 22.

    Gonzalez-Montalban N, Natalello A, Garcia-Fruitos E, Villaverde A, Doglia SM: In situ protein folding and activation in bacterial inclusion bodies. Biotechnol Bioeng. 2008, 100: 797-802. 10.1002/bit.21797.

    CAS  Article  Google Scholar 

  23. 23.

    de Marco A, Schroedel A: Characterization of the aggregates formed during recombinant protein expression in bacteria. BMC Biochem. 2005, 6: 10- 10.1186/1471-2091-6-10.

    Article  Google Scholar 

  24. 24.

    Martinez-Alonso M, Gonzalez-Montalban N, Garcia-Fruitos E, Villaverde A: The functional quality of soluble recombinant polypeptides produced in Escherichia coli is defined by a wide conformational spectrum. Appl Environ Microbiol. 2008

    Google Scholar 

  25. 25.

    Garcia-Fruitos E, Carrio MM, Aris A, Villaverde A: Folding of a misfolding-prone beta-galactosidase in absence of DnaK. Biotechnol Bioeng. 2005, 90: 869-875. 10.1002/bit.20496.

    CAS  Article  Google Scholar 

  26. 26.

    Gonzalez-Montalban N, Garcia-Fruitos E, Ventura S, Aris A, Villaverde A: The chaperone DnaK controls the fractioning of functional protein between soluble and insoluble cell fractions in inclusion body-forming cells. Microb Cell Fact. 2006, 5: 26- 10.1186/1475-2859-5-26.

    Article  Google Scholar 

  27. 27.

    Vallejo LF, Rinas U: Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fact. 2004, 3: 11- 10.1186/1475-2859-3-11.

    Article  Google Scholar 

  28. 28.

    Tsumoto K, Umetsu M, Kumagai I, Ejima D, Arakawa T: Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine. Biochem Biophys Res Commun. 2003, 312: 1383-1386. 10.1016/j.bbrc.2003.11.055.

    CAS  Article  Google Scholar 

  29. 29.

    Tsumoto K, Umetsu M, Kumagai I, Ejima D, Philo JS, Arakawa T: Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog. 2004, 20: 1301-1308. 10.1021/bp0498793.

    CAS  Article  Google Scholar 

  30. 30.

    Umetsu M, Tsumoto K, Nitta S, Adschiri T, Ejima D, Arakawa T, et al: Nondenaturing solubilization of beta2 microglobulin from inclusion bodies by L-arginine. Biochem Biophys Res Commun. 2005, 328: 189-197. 10.1016/j.bbrc.2004.12.156.

    CAS  Article  Google Scholar 

  31. 31.

    Peternel S, Grdadolnik J, Gaberc-Porekar V, Komel R: Engineering inclusion bodies for non denaturing extraction of functional proteins. Microb Cell Fact. 2008, 7: 34- 10.1186/1475-2859-7-34.

    Article  Google Scholar 

  32. 32.

    Peternel S, Jevsevar S, Bele M, Gaberc-Porekar V, Menart V: New properties of inclusion bodies with implications for biotechnology. Biotechnol Appl Biochem. 2008, 49: 239-246. 10.1042/BA20070140.

    CAS  Article  Google Scholar 

  33. 33.

    Jung KH: Enhanced enzyme activities of inclusion bodies of recombinant beta-galactosidase via the addition of inducer analog after L-arabinose induction in the araBAD promoter system of Escherichia coli. J Microbiol Biotechnol. 2008, 18: 434-442.

    CAS  Google Scholar 

  34. 34.

    Jung KH, Yeon JH, Moon SK, Choi JH: Methyl alpha-D-glucopyranoside enhances the enzymatic activity of recombinant beta-galactosidase inclusion bodies in the araBAD promoter system of Escherichia coli. J Ind Microbiol Biotechnol. 2008, 35: 695-701. 10.1007/s10295-008-0329-6.

    CAS  Article  Google Scholar 

  35. 35.

    Marston FA: The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J. 1986, 240: 1-12.

    CAS  Article  Google Scholar 

  36. 36.

    Sorensen HP, Mortensen KK: Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact. 2005, 4: 1- 10.1186/1475-2859-4-1.

    Article  Google Scholar 

  37. 37.

    Butt TR, Edavettal SC, Hall JP, Mattern MR: SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif. 2005, 43: 1-9. 10.1016/j.pep.2005.03.016.

    CAS  Article  Google Scholar 

  38. 38.

    Mansell TJ, Fisher AC, DeLisa MP: Engineering the protein folding landscape in gram-negative bacteria. Curr Protein Pept Sci. 2008, 9: 138-149. 10.2174/138920308783955243.

    CAS  Article  Google Scholar 

  39. 39.

    Wall JG, Pluckthun A: Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli. Curr Opin Biotechnol. 1995, 6: 507-516. 10.1016/0958-1669(95)80084-0.

    CAS  Article  Google Scholar 

  40. 40.

    de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B: Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol. 2007, 7: 32- 10.1186/1472-6750-7-32.

    Article  Google Scholar 

  41. 41.

    Sorensen HP, Mortensen KK: Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol. 2005, 115: 113-128. 10.1016/j.jbiotec.2004.08.004.

    CAS  Article  Google Scholar 

  42. 42.

    Nishihara K, Kanemori M, Yanagi H, Yura T: Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol. 2000, 66: 884-889. 10.1128/AEM.66.3.884-889.2000.

    CAS  Article  Google Scholar 

  43. 43.

    Song L, Yuan HJ, Coffey L, Doran J, Wang MX, Qian S, et al: Efficient expression in E. coli of an enantioselective nitrile hydratase from Rhodococcus erythropolis. Biotechnol Lett. 2008, 30: 755-762. 10.1007/s10529-007-9611-3.

    CAS  Article  Google Scholar 

  44. 44.

    de Marco A, De M V: Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning. J Biotechnol. 2004, 109: 45-52. 10.1016/j.jbiotec.2003.10.025.

    CAS  Article  Google Scholar 

  45. 45.

    Lee DH, Kim MD, Lee WH, Kweon DH, Seo JH: Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant Escherichia coli. Appl Microbiol Biotechnol. 2004, 63: 549-552. 10.1007/s00253-003-1370-z.

    CAS  Article  Google Scholar 

  46. 46.

    Liu D, Schmid RD, Rusnak M: Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm – a screening system for a frequently used biocatalyst. Appl Microbiol Biotechnol. 2006, 72: 1024-1032. 10.1007/s00253-006-0369-7.

    CAS  Article  Google Scholar 

  47. 47.

    Haacke A, Fendrich G, Ramage P, Geiser M: Chaperone over-expression in Escherichia coli: Apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates. Protein Expr Purif. 2008

    Google Scholar 

  48. 48.

    Kim SG, Kweon DH, Lee DH, Park YC, Seo JH: Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli. Protein Expr Purif. 2005, 41: 426-432. 10.1016/j.pep.2005.01.017.

    CAS  Article  Google Scholar 

  49. 49.

    Martinez-Alonso M, Garcia-Fruitos E, Villaverde A: Yield, solubility and conformational quality of soluble proteins are not simultaneously favored in recombinant Escherichia coli. Biotechnol Bioeng. 2008, 101: 1353-1358. 10.1002/bit.21996.

    CAS  Article  Google Scholar 

  50. 50.

    Carrio M, Gonzalez-Montalban N, Vera A, Villaverde A, Ventura S: Amyloid-like properties of bacterial inclusion bodies. J Mol Biol. 2005, 347: 1025-1037. 10.1016/j.jmb.2005.02.030.

    CAS  Article  Google Scholar 

  51. 51.

    Wang L, Maji SK, Sawaya MR, Eisenberg D, Riek R: Bacterial inclusion bodies contain amyloid-like structure. PLoS Biol. 2008, 6: e195- 10.1371/journal.pbio.0060195.

    Article  Google Scholar 

  52. 52.

    Speed MA, Wang DI, King J: Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition. Nat Biotechnol. 1996, 14: 1283-1287. 10.1038/nbt1096-1283.

    CAS  Article  Google Scholar 

  53. 53.

    Morell M, Bravo R, Espargaro A, Sisquella X, Aviles FX, Fernandez-Busquets X, et al: Inclusion bodies: specificity in their aggregation process and amyloid-like structure. Biochim Biophys Acta. 2008, 1783: 1815-1825. 10.1016/j.bbamcr.2008.06.007.

    CAS  Article  Google Scholar 

  54. 54.

    Rajan RS, Illing ME, Bence NF, Kopito RR: Specificity in intracellular protein aggregation and inclusion body formation. Proc Natl Acad Sci USA. 2001, 98: 13060-13065. 10.1073/pnas.181479798.

    CAS  Article  Google Scholar 

  55. 55.

    Jurgen B, Lin HY, Riemschneider S, Scharf C, Neubauer P, Schmid R, et al: Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations. Biotechnol Bioeng. 2000, 70: 217-224. 10.1002/1097-0290(20001020)70:2<217::AID-BIT11>3.0.CO;2-W.

    CAS  Article  Google Scholar 

  56. 56.

    Goff SA, Goldberg AL: Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell. 1985, 41: 587-595. 10.1016/S0092-8674(85)80031-3.

    CAS  Article  Google Scholar 

  57. 57.

    Allen SP, Polazzi JO, Gierse JK, Easton AM: Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli. J Bacteriol. 1992, 174: 6938-6947.

    CAS  Google Scholar 

  58. 58.

    Lesley SA, Graziano J, Cho CY, Knuth MW, Klock HE: Gene expression response to misfolded protein as a screen for soluble recombinant protein. Protein Eng. 2002, 15: 153-160. 10.1093/protein/15.2.153.

    CAS  Article  Google Scholar 

  59. 59.

    Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodriguez-Carmona E, Baumann K, et al: Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact. 2008, 7: 11- 10.1186/1475-2859-7-11.

    Article  Google Scholar 

Download references


We appreciate the financial support received for the design and production of recombinant proteins for biomedical applications from MEC (PETRI 95-0947.OP.02, BIO2005-23732-E, BIO2007-61194), AGAUR (2005SGR-00956), CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain, and from the European Science Foundation, which is also funded by the European Commission, Contract no. ERAS-CT-2003-980409 of the Sixth Framework Programme.

Author information



Corresponding author

Correspondence to Antonio Villaverde.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MMA, NGM and EGF have equally contributed to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Martínez-Alonso, M., González-Montalbán, N., García-Fruitós, E. et al. Learning about protein solubility from bacterial inclusion bodies. Microb Cell Fact 8, 4 (2009).

Download citation


  • Inclusion Body
  • Protein Species
  • Protein Version
  • Soluble Aggregate
  • Recombinant Protein Solubility