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

Nat Commun. 2013;4:2901. doi: 10.1038/ncomms3901.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biophysics / methods
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Directed Molecular Evolution
  • Endopeptidases / chemistry
  • Endopeptidases / genetics
  • Gene Library
  • High-Throughput Screening Assays / methods*
  • Interleukin 1 Receptor Antagonist Protein / chemistry
  • Interleukin 1 Receptor Antagonist Protein / genetics
  • Models, Molecular
  • Protein Engineering / methods*
  • Protein Stability*
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Surface Plasmon Resonance


  • IL1RN protein, human
  • Interleukin 1 Receptor Antagonist Protein
  • Single-Chain Antibodies
  • Endopeptidases
  • TEV protease

Associated data

  • PDB/4HVY