Efficient solubilization of inclusion bodies

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model‐protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was ∼75% while the fraction of soluble aggregates was ∼ 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.

[1]  Stephen P Bottomley,et al.  Protein expression and refolding--a practical guide to getting the most out of inclusion bodies. , 2004, Biotechnology annual review.

[2]  D. E. Anderson,et al.  pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme. , 1990, Biochemistry.

[3]  S. Singh,et al.  Solubilization and refolding of bacterial inclusion body proteins. , 2005, Journal of bioscience and bioengineering.

[4]  Antonio Villaverde A new editorial board for a new editorial period , 2004, Microbial cell factories.

[5]  P. Valax,et al.  Molecular Characterization of β‐Lactamase Inclusion Bodies Produced in Escherichia coli. 1. Composition , 1993, Biotechnology progress.

[6]  M. Brandon,et al.  Comparing the refolding and reoxidation of recombinant porcine growth hormone from a urea denatured state and from Escherichia coli inclusion bodies. , 1995, Biochemistry.

[7]  G. Makhatadze THERMODYNAMICS OF PROTEIN INTERACTIONS WITH UREA AND GUANIDINIUM HYDROCHLORIDE , 1999 .

[8]  E D Clark,et al.  Protein refolding for industrial processes. , 2001, Current opinion in biotechnology.

[9]  E Schwarz,et al.  Inhibition of aggregation side reactions during in vitro protein folding. , 1999, Methods in enzymology.

[10]  Alois Jungbauer,et al.  Current status of technical protein refolding. , 2007, Journal of biotechnology.

[11]  J. Chaudhuri,et al.  Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography. , 1999, Journal of biotechnology.

[12]  Charles Tanford,et al.  Isothermal Unfolding of Globular Proteins in Aqueous Urea Solutions , 1964 .

[13]  D. Segal,et al.  Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies. , 1995, Molecular immunology.

[14]  M. Yasuda,et al.  Effect of Additives on Refolding of a Denatured Protein , 1998, Biotechnology progress.

[15]  E. Kudryashova,et al.  Solubilization and refolding of inclusion body proteins in reverse micelles. , 2003, Analytical biochemistry.

[16]  A. Middelberg,et al.  Preparative protein refolding. , 2002, Trends in biotechnology.

[17]  R. Rudolph,et al.  Renaturation of human proinsulin--a study on refolding and conversion to insulin. , 2002, Analytical biochemistry.

[18]  Kouhei Tsumoto,et al.  Practical considerations in refolding proteins from inclusion bodies. , 2003, Protein expression and purification.

[19]  J. Chaudhuri,et al.  Refolding and purification of a urokinase plasminogen activator fragment by chromatography. , 2000, Journal of chromatography. B, Biomedical sciences and applications.

[20]  V. Uversky Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule. , 1993, Biochemistry.

[21]  Charles L. Cooney,et al.  Bioprocess simulation: An integrated approach to process development , 1989 .

[22]  Guifeng Zhang,et al.  Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. , 2002, Journal of chromatography. A.

[23]  B. Tang,et al.  Oxidative refolding of recombinant prochymosin. , 1999, The Biochemical journal.

[24]  Gail Sofer,et al.  Handbook of Process Chromatography: A Guide to Optimization, Scale Up, and Validation , 1997 .

[25]  T. Creighton Electrophoretic analysis of the unfolding of proteins by urea. , 1979, Journal of molecular biology.

[26]  K. Tsumoto,et al.  Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins , 2004, FEBS letters.

[27]  A. Patra,et al.  Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. , 2000, Protein expression and purification.

[28]  A. Panda,et al.  Solubilization of Recombinant Ovine Growth Hormone with Retention of Native‐like Secondary Structure and Its Refolding from the Inclusion Bodies of Escherichia coli , 1998, Biotechnology progress.

[29]  M. Pusey,et al.  Protein solubility modeling. , 1999, Biotechnology and bioengineering.

[30]  K. Inouye,et al.  Efficient solubilization, activation, and purification of recombinant Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli. , 2006, Protein expression and purification.

[31]  D. Ejima,et al.  Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine. , 2003, Biochemical and biophysical research communications.

[32]  Catherine H. Schein,et al.  Solubility as a Function of Protein Structure and Solvent Components , 1990, Bio/Technology.

[33]  Shigeki Nitta,et al.  Nondenaturing solubilization of beta2 microglobulin from inclusion bodies by L-arginine. , 2005, Biochemical and biophysical research communications.

[34]  Zhiguo Su,et al.  Refolding human lysozyme produced as an inclusion body by urea concentration and pH gradient ion exchange chromatography , 2002 .

[35]  Ursula Rinas,et al.  Microbial Cell Factories BioMed Central Review , 2003 .

[36]  Julian B. Chaudhuri,et al.  Refolding & Purification Of A Ukrinase Plasminogen Activator Fragment By Chromotography , 2001 .

[37]  R. Rudolph,et al.  In vitro folding of inclusion body proteins , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[38]  Z. Su,et al.  Urea gradient size-exclusion chromatography enhanced the yield of lysozyme refolding. , 2001, Journal of chromatography. A.

[39]  J. Chaudhuri,et al.  Considerations of sample application and elution during size-exclusion chromatography-based protein refolding. , 1999, Journal of chromatography. A.

[40]  Alois Jungbauer,et al.  Continuous matrix-assisted refolding of proteins. , 2003, Journal of chromatography. A.

[41]  J. Chaudhuri,et al.  Studies of the hydrodynamic volume changes that occur during refolding of lysozyme using size-exclusion chromatography. , 1997, Journal of chromatography. A.

[42]  Clark,et al.  Refolding of recombinant proteins , 1998, Current opinion in biotechnology.

[43]  The enthalpy of transfer of unfolded proteins into solutions of urea and guanidinium chloride. , 1996, Biophysical chemistry.