Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell

Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. Unfortunately, identifying stabilizing mutations is not trivial since most are neutral or deleterious. Here we describe a high-throughput colony-based stability screen, which is a direct and biophysical read-out of intrinsic protein stability in contrast to traditional indirect activity-based methods. By combining the method with a random mutagenesis procedure, we successfully identify thermostable variants from 10 diverse and challenging proteins, including several biotechnologically important proteins such as a single-chain antibody, a commercial enzyme and an FDA-approved protein drug. We also show that thermostabilization of a protein drug using our approach translates into dramatic improvements in long-term stability. As the method is generic and activity independent, it can easily be applied to a wide range of proteins.

[1]  P. Nordlund,et al.  Colony filtration blotting for screening soluble expression in Escherichia coli , 2006, Nature Protocols.

[2]  K. Colwill,et al.  A roadmap to generate renewable protein binders to the human proteome , 2011, Nature Methods.

[3]  P. Nordlund,et al.  Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination , 2006, Proceedings of the National Academy of Sciences.

[4]  G. Reeder,et al.  Development of a Stable Freeze-dried Formulation of Recombinant Human Interleukin-1 Receptor Antagonist , 1996, Pharmaceutical Research.

[5]  P. Hudson,et al.  Engineered antibody fragments and the rise of single domains , 2005, Nature Biotechnology.

[6]  V. Eijsink,et al.  Directed evolution of enzyme stability. , 2005, Biomolecular engineering.

[7]  F. Cava,et al.  An activity-independent selection system of thermostable protein variants , 2007, Nature Methods.

[8]  Ursula Egner,et al.  Identifying protein construct variants with increased crystallization propensity––A case study , 2006, Protein science : a publication of the Protein Society.

[9]  V. Eijsink,et al.  Selection of mutations for increased protein stability. , 2002, Current opinion in biotechnology.

[10]  Jijun Hao,et al.  A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents. , 2004, Protein engineering, design & selection : PEDS.

[11]  Joseph D. Kwasnoski,et al.  High-density miniaturized thermal shift assays as a general strategy for drug discovery. , 2001, Journal of biomolecular screening.

[12]  Masoud Vedadi,et al.  Screening for Ligands Using a Generic and High-Throughput Light-Scattering-Based Assay , 2006, Journal of biomolecular screening.

[13]  K. Miyazaki.  Creating random mutagenesis libraries by megaprimer PCR of whole plasmid (MEGAWHOP). , 2003, Methods in molecular biology.

[14]  K. Dill,et al.  The Protein-Folding Problem, 50 Years On , 2012, Science.

[15]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[16]  S. Radford,et al.  Optimizing protein stability in vivo. , 2009, Molecular cell.

[17]  Christopher G Tate,et al.  Development and crystallization of a minimal thermostabilised G protein-coupled receptor. , 2009, Protein expression and purification.

[18]  Christopher J. Oldfield,et al.  Do viral proteins possess unique biophysical features? , 2009, Trends in biochemical sciences.

[19]  Naomi E Chayen,et al.  Protein crystallization: from purified protein to diffraction-quality crystal , 2008, Nature Methods.

[20]  H A Schreuder,et al.  Refined crystal structure of the interleukin-1 receptor antagonist. Presence of a disulfide link and a cis-proline. , 1995, European journal of biochemistry.

[21]  P. D. de Jong,et al.  Ligation-independent cloning of PCR products (LIC-PCR). , 1990, Nucleic acids research.

[22]  George T Detitta,et al.  Thermofluor-based high-throughput stability optimization of proteins for structural studies. , 2006, Analytical biochemistry.

[23]  F. Arnold,et al.  Protein stability promotes evolvability. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Aditya A Wakankar,et al.  Physicochemical stability of the antibody-drug conjugate Trastuzumab-DM1: changes due to modification and conjugation processes. , 2010, Bioconjugate chemistry.

[25]  A. Plückthun,et al.  Stability engineering of antibody single-chain Fv fragments. , 2001, Journal of molecular biology.

[26]  Claire Dumon,et al.  Engineering Hyperthermostability into a GH11 Xylanase Is Mediated by Subtle Changes to Protein Structure* , 2008, Journal of Biological Chemistry.

[27]  S. Krishnan,et al.  Multistep aggregation pathway of human interleukin-1 receptor antagonist: kinetic, structural, and morphological characterization. , 2009, Biophysical journal.

[28]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[29]  Helena Berglund,et al.  Improved solubility of TEV protease by directed evolution. , 2006, Journal of biotechnology.

[30]  Huub Schellekens,et al.  Bioequivalence and the immunogenicity of biopharmaceuticals , 2002, Nature Reviews Drug Discovery.

[31]  Randal R Ketchem,et al.  Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist. , 2007, Journal of molecular biology.

[32]  F. Castellino,et al.  A differential scanning calorimetric investigation of the domains of recombinant tissue plasminogen activator. , 1988, Archives of biochemistry and biophysics.

[33]  Anthony Mire-Sluis,et al.  Characterizing biological products and assessing comparability following manufacturing changes , 2004, Nature Biotechnology.

[34]  A. Plückthun,et al.  High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. , 1999, Cancer research.

[35]  M. Lehmann,et al.  Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution. , 2001, Current opinion in biotechnology.

[36]  A. Plückthun,et al.  Selecting proteins with improved stability by a phage-based method , 1998, Nature Biotechnology.

[37]  Ronald W. Barrett,et al.  A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist , 1997, Nature.

[38]  D. Dimitrov.  Engineered CH2 domains (nanoantibodies) , 2009, mAbs.

[39]  I. Kurnaz.  Protein Production and Purification , 2015 .

[40]  Pär Nordlund,et al.  Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli , 2005, Nature Methods.

[41]  S. Frokjaer,et al.  Thermal dissociation and unfolding of insulin. , 2005, Biochemistry.

[42]  Rajan Sankaranarayanan,et al.  Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight. , 2008, Journal of molecular biology.

[43]  Gebhard F. X. Schertler,et al.  Structure of a β1-adrenergic G-protein-coupled receptor , 2008, Nature.

[44]  Bruce A Kerwin,et al.  Anion binding and controlled aggregation of human interleukin-1 receptor antagonist. , 2005, Biochemistry.

[45]  F. Arnold,et al.  Directed evolution of a thermostable esterase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[46]  C. J. Bond,et al.  Comprehensive Analysis of the Factors Contributing to the Stability and Solubility of Autonomous Human VH Domains* , 2008, Journal of Biological Chemistry.

[47]  Robert Gurny,et al.  Stability of human growth hormone: influence of methionine oxidation on thermal folding. , 2011, Journal of pharmaceutical sciences.

[48]  S. Rybak,et al.  Generation of a highly stable, internalizing anti‐CD22 single‐chain Fv fragment for targeting non‐Hodgkin's lymphoma , 2003, International journal of cancer.