Post-production protein stability: trouble beyond the cell factory
Microbial Cell Factories volume 10, Article number: 60 (2011)
Being protein function a conformation-dependent issue, avoiding aggregation during production is a major challenge in biotechnological processes, what is often successfully addressed by convenient upstream, midstream or downstream approaches. Even when obtained in soluble forms, proteins tend to aggregate, especially if stored and manipulated at high concentrations, as is the case of protein drugs for human therapy. Post-production protein aggregation is then a major concern in the pharmaceutical industry, as protein stability, pharmacokinetics, bioavailability, immunogenicity and side effects are largely dependent on the extent of aggregates formation. Apart from acting at the formulation level, the recombinant nature of protein drugs allows intervening at upstream stages through protein engineering, to produce analogue protein versions with higher stability and enhanced therapeutic values.
Aggregation and associated conformational stress of cell factories (both prokaryotic and eukaryotic) are major concerns in recombinant protein production, resulting in low yields, unstable production and limited solubility and biological activity of the products [1–9]. Basic research on protein folding and the routine implementation of several analytical procedures such as circular dichroism, mass spectrometry and infrared spectroscopy (mostly incorporated from amyloid research) [4, 10–16] have expanded our understanding of how polypeptide chains cross-interact and aggregate in vivo. In bacteria, probably the most studied cell factories, aggregation as inclusion bodies, a quite common event during production of heterologous polypeptides [17, 18], is now observed as a complex physiological event in which cellular agents, including chaperones [6, 19, 20], proteases [21–23] and actin-like proteins  are coordinately acting [24, 25] in the frame of the cell's protein quality control machinery [26–28]. Despite aggregation as inclusion bodies might represent a source of relatively pure proteins for further refolding or extraction [29–33], or unexpectedly, a new type of nano-microparticulate biomaterials for biotechnological and biomedical applications [34–39], the use of recombinant proteins for most of biotechnological and biomedical applications requires fully soluble protein versions. A particular issue in recombinant protein aggregation is the occurrence of soluble aggregates (less apparent that large aggregates), that are being progressively recognized in production processes. These soluble clusters adopt a spectrum of forms (mainly fibrilar, spherical or amorphous) [40, 41] and might be the in vivo physiological precursors and structural components of bacterial inclusion bodies [24, 42]. Very different approaches have been explored at upstream, midstream and downstream levels to minimize aggregation during recombinant protein production (Figure 1). Such strategies, eventhough being mostly a trial-and-error process, often result in significant improvements of protein solubility [43–46].
Desirably, soluble versions of recombinant proteins should keep such soluble status in post-production stages, that is, during storage and use. This need is specially acute in the case of proteins intended for therapeutic uses [47, 48]. Protein drugs are commonly administered parenterally , what makes protein aggregation in stocks or upon administration a main concern in the Pharma industry (see for instance, http://www.eahp.eu/content/download/25193/164355/.../CoverStory20-21.pdf.pd). The high concentration at which proteins drugs are stored and administered  specifically favors aggregation . In this context, diverse analytical procedures have been developed and specifically adapted to the detection of therapeutic protein aggregation [52–54]. Importantly, aggregation does not only render drug inactivation during storage, and fast clearance, reduction of activity, limited bioavailability and proteolytic digestion upon administration, but it also stimulates undesired immunogenicity . This is a critical issue in clinics as severe side effects observed upon prolonged protein administration (as in the case of insulins, interferons, erythropoietin and growth hormone) are antibody-dependent [56–59].
Chemical modification of proteins and the use of appropriate excipients (Figure 1) are the most taken approaches for protein drug stabilization [60–62]. Obviously, emerging concepts in Nanotechnology, Nanomedicine and in Material Sciences offer new biocompatible vehicles for protein encapsulation or embedding, mainly at the nanoscale, through which the stability, tissue targeting and bioavailability during drug delivery are dramatically enhanced [63, 64]. Many among those such nanostructured materials are from bacterial origin .
Being proteins flexible molecules suitable to be re-designed by genetic methods, upstream protein engineering, one of the main approaches to prevent aggregation during production (Table 1, up), is also useful to stabilize protein drugs during use (Table 1, bottom). In addition, modification of the protein primary sequence permits a fine tuning of protein features such as oligomerization, activity, cell targeting and cell penetration, that represent additional values in the performance of a protein drug (Table 1; Figure 1, bottom). In this regard, protein engineering is revealed as an extremely flexible approach to enhance the stability of proteins during production, storage and use, but also to improve their performance in in vivo uses. Reduction of aggregation is expected to minimize immunogenicity, increase proteolytic stability, improve bioavailability and limit side-effects, as aggregation has a pivotal role in all these issues [55, 59, 66–69]. In addition, protein modification can offer added values to protein drugs, by conferring novel functions that improve pharmacological performance without necessarily enhancing solubility (Figure 1, green framed box). These include cell or tissue targeting or enhanced cell penetration by the fusion to a cell receptor ligand or an antibody [70–74], enhancing half-life and bioavailability by fusion to transferrin , albumin , or albumin-binding peptides  and crossing the brain-blood barrier (BBB) by the incorporation of cationic peptides . Creating multifunctional proteins by the appropriate combination of protein domains in a single polypeptide chain is being especially explored for the construction of protein-only artificial viruses, in which the therapeutic nucleic acids are encapsulated by chimerical protein building blocks [72, 79–81]. Further exploration of protein engineering focused on post-production issues is strongly required and it should allow the emergence of optimized drugs to fulfill their increasing demand.
Stability and solubility of recombinant proteins is a critical issue at both production and post-production stages. For a biomedical use of proteins as pharmaceuticals, high solubility not only supports stability but it also enhances bioavailability and reduces immunogenicity and undesired toxic effects. Among other approaches to stabilize protein drugs, such as chemical modification, proper formulation and encapsulation, protein engineering is a very flexible route to improve protein folding during production and reduce aggregation during storage and in vivo. Furthermore, the modification of protein primary sequence permits to confer additional functional values, such as binding to serum albumin, binding to cell surface receptors and cell membrane (or BBB) crossing, thus improving biodistribution, expanding the half-life and enhancing the biological potential of the drug. The wide spectrum of possibilities of post-production-addressed protein engineering is probably to be yet realized.
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.
Ciplys E, Samuel D, Juozapaitis M, Sasnauskas K, Slibinskas R: Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae. Microb Cell Fact. 2011, 10: 37-10.1186/1475-2859-10-37.
Parrilli E, Giuliani M, Marino G, Tutino ML: Influence of production process design on inclusion bodies protein: the case of an Antarctic flavohemoglobin. Microb Cell Fact. 2010, 9: 19-10.1186/1475-2859-9-19.
Ami D, Natalello A, Schultz T, Gatti-Lafranconi P, Lotti M, Doglia SM, et al: Effects of recombinant protein misfolding and aggregation on bacterial membranes. Biochim Biophys Acta. 2009, 1794: 263-269.
Guillemette T, van Peij NN, Goosen T, Lanthaler K, Robson GD, Van Den Hondel CA, et al: Genomic analysis of the secretion stress response in the enzyme-producing cell factory Aspergillus niger. BMC Genomics. 2007, 8: 158-10.1186/1471-2164-8-158.
Martinez-Alonso M, Garcia-Fruitos E, Ferrer-Miralles N, Rinas U, Villaverde A: Side effects of chaperone gene co-expression in recombinant protein production. Microb Cell Fact. 2010, 9: 64-10.1186/1475-2859-9-64.
Martinez-Alonso M, Gonzalez-Montalban N, Garcia-Fruitos E, Villaverde A: Learning about protein solubility from bacterial inclusion bodies. Microb Cell Fact. 2009, 8: 4-10.1186/1475-2859-8-4.
Dragosits M, Frascotti G, Bernard-Granger L, Vazquez F, Giuliani M, Baumann K, et al: Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis. Biotechnol Prog. 2011, 27: 38-46. 10.1002/btpr.524.
Mattanovich D, Gasser B, Hohenblum H, Sauer M: Stress in recombinant protein producing yeasts. J Biotechnol. 2004, 113: 121-135. 10.1016/j.jbiotec.2004.04.035.
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.
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.
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.
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.
Ami D, Natalello A, Taylor G, Tonon G, Maria DS: Structural analysis of protein inclusion bodies by Fourier transform infrared microspectroscopy. Biochim Biophys Acta. 2006, 1764: 793-799.
Ami D, Natalello A, Gatti-Lafranconi P, Lotti M, Doglia SM: Kinetics of inclusion body formation studied in intact cells by FT-IR spectroscopy. FEBS Lett. 2005, 579: 3433-3436. 10.1016/j.febslet.2005.04.085.
Ami D, Bonecchi L, Cali S, Orsini G, Tonon G, Doglia SM: FT-IR study of heterologous protein expression in recombinant Escherichia coli strains. Biochim Biophys Acta. 2003, 1624: 6-10.
Villaverde A, Carrio MM: Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett. 2003, 25: 1385-1395. 10.1023/A:1025024104862.
Baneyx F, Mujacic M: Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol. 2004, 22: 1399-1408. 10.1038/nbt1029.
Gonzalez-Montalban N, Carrio MM, Cuatrecasas S, Aris A, Villaverde A: Bacterial inclusion bodies are cytotoxic in vivo in absence of functional chaperones DnaK or GroEL. J Biotechnol. 2005, 118 (4): 406-412.
Carrio MM, Villaverde A: Localization of chaperones DnaK and GroEL in bacterial inclusion bodies. J Bacteriol. 2005, 187: 3599-3601. 10.1128/JB.187.10.3599-3601.2005.
Jurgen B, Breitenstein A, Urlacher V, Buttner K, Lin H, Hecker M, et al: Quality control of inclusion bodies in Escherichia coli. Microb Cell Fact. 2010, 9: 41-10.1186/1475-2859-9-41.
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.
Carbonell X, Villaverde A: Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion. Biotechnology Letters. 2002, 24: 1939-1944. 10.1023/A:1021173114193.
Rokney A, Shagan M, Kessel M, Smith Y, Rosenshine I, Oppenheim AB: E. coli transports aggregated proteins to the poles by a specific and energy-dependent process. J Mol Biol. 2009, 392: 589-601. 10.1016/j.jmb.2009.07.009.
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.
Ventura S, Villaverde A: Protein quality in bacterial inclusion bodies. Trends Biotechnol. 2006, 24: 179-185. 10.1016/j.tibtech.2006.02.007.
Tyedmers J, Mogk A, Bukau B: Cellular strategies for controlling protein aggregation. Nat Rev Mol Cell Biol. 2010, 11 (11): 777-788.
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.
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.
Jungbauer A, Kaar W: Current status of technical protein refolding. J Biotechnol. 2007, 128: 587-596. 10.1016/j.jbiotec.2006.12.004.
Singh SM, Panda AK: Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng. 2005, 99: 303-310. 10.1263/jbb.99.303.
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.
Cabrita LD, Bottomley SP: Protein expression and refolding - A practical guide to getting the most out of inclusion bodies. Biotechnol Annu Rev. 2004, 10: 31-50.
Garcia-Fruitos E, Villaverde A: Friendly production of bacterial inclusion bodies. Korean J Chem Eng. 2010, 27: 385-389.
Vazquez E, Villaverde A: Engineering building blocks for self-assembling protein nanoparticles. Microb Cell Fact. 2010, 9: 101-10.1186/1475-2859-9-101.
Villaverde A: Nanotechnology, bionanotechnology and microbial cell factories. Microb Cell Fact. 2010, 9: 53-10.1186/1475-2859-9-53.
Rodriguez-Carmona E, Cano-Garrido O, Seras-Franzoso J, Villaverde A, Garcia-Fruitos E: Isolation of cell-free bacterial inclusion bodies. Microb Cell Fact. 2010, 9: 71-10.1186/1475-2859-9-71.
Peternel S, Komel R: Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells. Microb Cell Fact. 2010, 9: 66-10.1186/1475-2859-9-66.
Garcia-Fruitos E: Inclusion bodies: a new concept. Microb Cell Fact. 2010, 9: 80-10.1186/1475-2859-9-80.
de Marco A, Sevastsyanovich YR, Cole JA: Minimal information for protein functional evaluation (MIPFE) workshop. N Biotechnol. 2009, 25: 170-10.1016/j.nbt.2008.12.006.
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.
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.
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.
Makino T, Skretas G, Georgiou G: Strain engineering for improved expression of recombinant proteins in bacteria. Microb Cell Fact. 2011, 10: 32-10.1186/1475-2859-10-32.
Kolaj O, Spada S, Robin S, Wall JG: Use of folding modulators to improve heterologous protein production in Escherichia coli. Microb Cell Fact. 2009, 8: 9-10.1186/1475-2859-8-9.
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.
Ferrer-Miralles N, Domingo-Espin J, Corchero JL, Vazquez E, Villaverde A: Microbial factories for recombinant pharmaceuticals. Microb Cell Fact. 2009, 8: 17-10.1186/1475-2859-8-17.
Leader B, Baca QJ, Golan DE: Protein therapeutics: a summary and pharmacological classification. Nat Rev Drug Discov. 2008, 7: 21-39. 10.1038/nrd2399.
Antosova Z, Mackova M, Kral V, Macek T: Therapeutic application of peptides and proteins: parenteral forever?. Trends Biotechnol. 2009, 27: 628-635. 10.1016/j.tibtech.2009.07.009.
Shire SJ, Shahrokh Z, Liu J: Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004, 93: 1390-1402. 10.1002/jps.20079.
Ellis RJ, Minton AP: Protein aggregation in crowded environments. Biol Chem. 2006, 387: 485-497. 10.1515/BC.2006.064.
Garcia-Fruitos E, Vazquez E, Gonzalez-Montalban N, Ferrer-Miralles N, Villaverde A: Analytical Approaches for Assessing Aggregation of Protein Biopharmaceuticals. Curr Pharm Biotechnol. 2011
Bobst CE, Kaltashov IA: Advanced Mass Spectrometry-Based Methods for the Analysis of Conformational Integrity of Biopharmaceutical Products. Curr Pharm Biotechnol. 2011
Bertucci C, Pistolozzi M, De SA: Structural Characterization of Recombinant Therapeutic Proteins by Circular Dichroism. Curr Pharm Biotechnol. 2011
Richard J, Prang N: The formulation and immunogenicity of therapeutic proteins: Product quality as a key factor. IDrugs. 2010, 13: 550-558.
Katagiri D, Shibata M, Katsuki T, Masumoto S, Katsuma A, Minami E, et al: Antiepoetin antibody-related pure red cell aplasia: successful remission with cessation of recombinant erythropoietin alone. Clin Exp Nephrol. 2010, 14: 501-505. 10.1007/s10157-010-0318-8.
Braun A, Kwee L, Labow MA, Alsenz J: Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice. Pharm Res. 1997, 14: 1472-1478. 10.1023/A:1012193326789.
Frost H: Antibody-mediated side effects of recombinant proteins. Toxicology. 2005, 209: 155-160. 10.1016/j.tox.2004.12.028.
Fradkin AH, Carpenter JF, Randolph TW: Immunogenicity of aggregates of recombinant human growth hormone in mouse models. J Pharm Sci. 2009, 98: 3247-3264. 10.1002/jps.21834.
Jorgensen L, Hostrup S, Moeller EH, Grohganz H: Recent trends in stabilising peptides and proteins in pharmaceutical formulation - considerations in the choice of excipients. Expert Opin Drug Deliv. 2009, 6: 1219-1230. 10.1517/17425240903199143.
Arakawa T, Tsumoto K, Kita Y, Chang B, Ejima D: Biotechnology applications of amino acids in protein purification and formulations. Amino Acids. 2007, 33: 587-605. 10.1007/s00726-007-0506-3.
Frokjaer S, Otzen DE: Protein drug stability: a formulation challenge. Nat Rev Drug Discov. 2005, 4: 298-306. 10.1038/nrd1695.
Parveen S, Misra R, Sahoo SK: Nanoparticles: a boon to drug delivery, therapeutics, diagnostics and imaging. Nanomedicine. 2011
Singh S: Nanomedicine-nanoscale drugs and delivery systems. J Nanosci Nanotechnol. 2010, 10: 7906-7918. 10.1166/jnn.2010.3617.
Rodriguez-Carmona E, Villaverde A: Nanostructured bacterial materials for innovative medicines. Trends Microbiol. 2010, 18: 423-430. 10.1016/j.tim.2010.06.007.
Antonelli G: Reflections on the immunogenicity of therapeutic proteins. Clin Microbiol Infect. 2008, 14: 731-733. 10.1111/j.1469-0691.2008.01969.x.
De Groot AS, Scott DW: Immunogenicity of protein therapeutics. Trends Immunol. 2007, 28: 482-490. 10.1016/j.it.2007.07.011.
Hermeling S, Schellekens H, Maas C, Gebbink MF, Crommelin DJ, Jiskoot W: Antibody response to aggregated human interferon alpha2b in wild-type and transgenic immune tolerant mice depends on type and level of aggregation. J Pharm Sci. 2006, 95: 1084-1096. 10.1002/jps.20599.
Schellekens H: Bioequivalence and the immunogenicity of biopharmaceuticals. Nat Rev Drug Discov. 2002, 1: 457-462. 10.1038/nrd818.
Pardridge WM: Blood-brain barrier delivery of protein and non-viral gene therapeutics with molecular Trojan horses. J Control Release. 2007, 122 (3): 345-348.
Sondel PM, Hank JA, Gan J, Neal Z, Albertini MR: Preclinical and clinical development of immunocytokines. Curr Opin Investig Drugs. 2003, 4: 696-700.
Vazquez E, Ferrer-Miralles N, Mangues R, Corchero JL, Schwartz S, Villaverde A: Modular protein engineering in emerging cancer therapies. Curr Pharm Des. 2009, 15: 893-916. 10.2174/138161209787582084.
Boado RJ, Zhang Y, Zhang Y, Pardridge WM: Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier. Biotechnol Bioeng. 2007, 97: 1376-1386. 10.1002/bit.21369.
Dietz GP, Bahr M: Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol Cell Neurosci. 2004, 27: 85-131. 10.1016/j.mcn.2004.03.005.
Amet N, Wang W, Shen WC: Human growth hormone-transferrin fusion protein for oral delivery in hypophysectomized rats. J Control Release. 2010, 141: 177-182. 10.1016/j.jconrel.2009.09.007.
Subramanian GM, Fiscella M, Lamouse-Smith A, Zeuzem S, McHutchison JG: Albinterferon alpha-2b: a genetic fusion protein for the treatment of chronic hepatitis C. Nat Biotechnol. 2007, 25: 1411-1419. 10.1038/nbt1364.
Dennis MS, Zhang M, Meng YG, Kadkhodayan M, Kirchhofer D, Combs D, et al: Albumin binding as a general strategy for improving the pharmacokinetics of proteins. J Biol Chem. 2002, 277: 35035-35043. 10.1074/jbc.M205854200.
de Boer AG, Gaillard PJ: Drug targeting to the brain. Annu Rev Pharmacol Toxicol. 2007, 47: 323-355. 10.1146/annurev.pharmtox.47.120505.105237.
Aris A, Villaverde A: Modular protein engineering for non-viral gene therapy. Trends Biotechnol. 2004, 22: 371-377. 10.1016/j.tibtech.2004.05.004.
Vazquez E, Ferrer-Miralles N, Villaverde A: Peptide-assisted traffic engineering for nonviral gene therapy. Drug Discov Today. 2008, 13: 1067-1074. 10.1016/j.drudis.2008.08.008.
Ferrer-Miralles N, Vazquez E, Villaverde A: Membrane-active peptides for non-viral gene therapy: making the safest easier. Trends Biotechnol. 2008, 26: 267-275. 10.1016/j.tibtech.2008.02.003.
Rosano GL, Ceccarelli EA: Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microb Cell Fact. 2009, 8: 41-10.1186/1475-2859-8-41.
Lee Y, Zhou T, Tartaglia GG, Vendruscolo M, Wilke CO: Translationally optimal codons associate with aggregation-prone sites in proteins. Proteomics. 2010, 10: 4163-4171. 10.1002/pmic.201000229.
Kamionka M: Engineering of therapeutic proteins production in Escherichia coli. Curr Pharm Biotechnol. 2011, 12: 268-274. 10.2174/138920111794295693.
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.
Strandberg L, Enfors SO: Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli. Appl Environ Microbiol. 1991, 57: 1669-1674.
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.
de Marco A: Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli. Nat Protoc. 2007, 2: 2632-2639. 10.1038/nprot.2007.400.
de Marco A: Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli. Nat Protoc. 2007, 2: 2632-2639. 10.1038/nprot.2007.400.
Makrides SC: Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev. 1996, 60: 512-538.
Gleeson MA, White CE, Meininger DP, Komives EA: Generation of protease-deficient strains and their use in heterologous protein expression. Methods Mol Biol. 1998, 103: 81-94.
de Marco A: Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb Cell Fact. 2009, 8: 26-10.1186/1475-2859-8-26.
Gibson TJ, Mccarty K, Mcfadyen IJ, Cash E, Dalmonte P, Hinds KD, et al: Application of a high-throughput screening procedure with PEG-induced precipitation to compare relative protein solubility during formulation development with IgG1 monoclonal antibodies. J Pharm Sci. 2011, 100: 1009-1021. 10.1002/jps.22350.
Bondos SE, Bicknell A: Detection and prevention of protein aggregation before, during, and after purification. Anal Biochem. 2003, 316: 223-231. 10.1016/S0003-2697(03)00059-9.
Golovanov AP, Hautbergue GM, Wilson SA, Lian LY: A simple method for improving protein solubility and long-term stability. J Am Chem Soc. 2004, 126: 8933-8939. 10.1021/ja049297h.
Ducat T, Declerck N, Gostan T, Kochoyan M, Demene H: Rapid determination of protein solubility and stability conditions for NMR studies using incomplete factorial design. J Biomol NMR. 2006, 34: 137-151. 10.1007/s10858-006-0003-0.
Gosavi RA, Mueser TC, Schall CA: Optimization of buffer solutions for protein crystallization. Acta Crystallogr D Biol Crystallogr. 2008, 64: 506-514. 10.1107/S0907444908004265.
Rathore N, Rajan RS: Current perspectives on stability of protein drug products during formulation, fill and finish operations. Biotechnol Prog. 2008, 24: 504-514. 10.1021/bp070462h.
Tiwari AK, Gajbhiye V, Sharma R, Jain NK: Carrier mediated protein and peptide stabilization. Drug Deliv. 2010, 17: 605-616. 10.3109/10717544.2010.509359.
Rinas U, Tsai LB, Lyons D, Fox GM, Stearns G, Fieschko J, et al: Cysteine to serine substitutions in basic fibroblast growth factor: effect on inclusion body formation and proteolytic susceptibility during in vitro refolding. Biotechnology NY. 1992, 10: 435-440. 10.1038/nbt0492-435.
Lawson AJ, Walker EA, White SA, Dafforn TR, Stewart PM, Ride JP: Mutations of key hydrophobic surface residues of 11 beta-hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system. Protein Sci. 2009, 18: 1552-1563. 10.1002/pro.150.
Li P, Guan H, Li J, Lin Z: Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli. Protein Expr Purif. 2009, 65: 196-203. 10.1016/j.pep.2008.11.012.
Kong B, Guo GL: Enhanced In Vitro Refolding of Fibroblast Growth Factor 15 with the Assistance of SUMO Fusion Partner. PLoS One. 2011, 6: e20307-10.1371/journal.pone.0020307.
Jung HJ, Kim SK, Min WK, Lee SS, Park K, Park YC, et al: Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli. Bioprocess Biosyst Eng. 2011
Kato A, Maki K, Ebina T, Kuwajima K, Soda K, Kuroda Y: Mutational analysis of protein solubility enhancement using short peptide tags. Biopolymers. 2007, 85: 12-18. 10.1002/bip.20596.
Guo W, Cao L, Jia Z, Wu G, Li T, Lu F, et al: High level soluble production of functional ribonuclease inhibitor in Escherichia coli by fusing it to soluble partners. Protein Expr Purif. 2011, 77: 185-192. 10.1016/j.pep.2011.01.015.
Davis GD, Elisee C, Newham DM, Harrison RG: New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnol Bioeng. 1999, 65: 382-388. 10.1002/(SICI)1097-0290(19991120)65:4<382::AID-BIT2>3.0.CO;2-I.
De M, Stier G, Blandin S, de MA: The solubility and stability of recombinant proteins are increased by their fusion to NusA. Biochem Biophys Res Commun. 2004, 322: 766-771. 10.1016/j.bbrc.2004.07.189.
Mudaliar SR, Lindberg FA, Joyce M, Beerdsen P, Strange P, Lin A, et al: Insulin aspart (B28 asp-insulin): a fast-acting analog of human insulin: absorption kinetics and action profile compared with regular human insulin in healthy nondiabetic subjects. Diabetes Care. 1999, 22: 1501-1506. 10.2337/diacare.22.9.1501.
O'Rourke EC, Drummond RJ, Creasey AA: Binding of 125I-labeled recombinant beta interferon (IFN-beta Ser17) to human cells. Mol Cell Biol. 1984, 4: 2745-2749.
Hsu E, Osslund T, Nybo R, Chen BL, Kenney WC, Morris CF, et al: Enhanced stability of recombinant keratinocyte growth factor by mutagenesis. Protein Eng Des Sel. 2006, 19: 147-153. 10.1093/protein/gzj013.
Blatt LM, Davis JM, Klein SB, Taylor MW: The biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon. J Interferon Cytokine Res. 1996, 16: 489-499. 10.1089/jir.1996.16.489.
Justesen S, Buus S, Claesson MH, Pedersen AE: Addition of TAT protein transduction domain and GrpE to human p53 provides soluble fusion proteins that can be transduced into dendritic cells and elicit p53-specific T-cell responses in HLA-A*0201 transgenic mice. Immunology. 2007, 122: 326-334. 10.1111/j.1365-2567.2007.02643.x.
Harada H, Hiraoka M, Kizaka-Kondoh S: Antitumor effect of TAT-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumor cells. Cancer Res. 2002, 62: 2013-2018.
Vocero-Akbani AM, Heyden NV, Lissy NA, Ratner L, Dowdy SF: Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. Nat Med. 1999, 5: 29-33. 10.1038/4710.
We appreciate the financial support received for the design and microbial production of recombinant proteins for biomedical applications from FISS (PS09/00165), MICINN (BFU2010-17450, ACI2009-0919, IT2009-0021, EUI2008-03610), AGAUR (2009SGR-108), 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 (ERANET-IB 08-007). The authors also appreciate the financial support through the project "Development of nanomedicines for enzymatic replacement therapy in Fabry disease" granted by the Fundació Marató TV3.
The authors declare that they have no competing interests.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Vazquez, E., Corchero, J.L. & Villaverde, A. Post-production protein stability: trouble beyond the cell factory. Microb Cell Fact 10, 60 (2011). https://doi.org/10.1186/1475-2859-10-60
- Protein Engineering
- Recombinant Protein Production
- Protein Drug
- Protein Quality Control
- Protein Version