• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of prosciprotein sciencecshl presssubscriptionsetoc alertsthe protein societyjournal home
Protein Sci. Sep 1999; 8(9): 1908–1911.
PMCID: PMC2144404

A simple in vivo assay for increased protein solubility.


Low solubility is a major stumbling block in the detailed structural and functional characterization of many proteins and isolated protein domains. The production of some proteins in a soluble form may only be possible through alteration of their sequences by mutagenesis. The feasibility of this approach has been demonstrated in a number of cases where amino acid substitutions were shown to increase protein solubility without altering structure or function. However, identifying residues to mutagenize to increase solubility is difficult, especially in the absence of structural knowledge. For this reason, we have developed a method by which soluble mutants of an insoluble protein can be easily distinguished in vivo in Escherichia coli. This method is based on our observation that cells expressing fusions of an insoluble protein to chloramphenicol acetyltransferase (CAT) exhibit decreased resistance to chloramphenicol compared to fusions with soluble proteins. We found that a soluble mutant of an insoluble protein fused to CAT could be selected by plating on high levels of chloramphenicol.

Full Text

The Full Text of this article is available as a PDF (98K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Dale GE, Broger C, Langen H, D'Arcy A, Stüber D. Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase. Protein Eng. 1994 Jul;7(7):933–939. [PubMed]
  • Dyda F, Hickman AB, Jenkins TM, Engelman A, Craigie R, Davies DR. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science. 1994 Dec 23;266(5193):1981–1986. [PubMed]
  • Jenkins TM, Hickman AB, Dyda F, Ghirlando R, Davies DR, Craigie R. Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6057–6061. [PMC free article] [PubMed]
  • Ko YH, Thomas PJ, Delannoy MR, Pedersen PL. The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein. J Biol Chem. 1993 Nov 15;268(32):24330–24338. [PubMed]
  • Leistler B, Perham RN. Solubilizing buried domains of proteins: a self-assembling interface domain from glutathione reductase. Biochemistry. 1994 Mar 15;33(10):2773–2781. [PubMed]
  • Murby M, Samuelsson E, Nguyen TN, Mignard L, Power U, Binz H, Uhlén M, Ståhl S. Hydrophobicity engineering to increase solubility and stability of a recombinant protein from respiratory syncytial virus. Eur J Biochem. 1995 May 15;230(1):38–44. [PubMed]
  • Nieba L, Honegger A, Krebber C, Plückthun A. Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng. 1997 Apr;10(4):435–444. [PubMed]
  • Pan H, Clary D, Sadowski PD. Identification of the DNA-binding domain of the FLP recombinase. J Biol Chem. 1991 Jun 15;266(17):11347–11354. [PubMed]
  • Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. [PubMed]
  • Robben J, Massie G, Bosmans E, Wellens B, Volckaert G. An Escherichia coli plasmid vector system for high-level production and purification of heterologous peptides fused to active chloramphenicol acetyltransferase. Gene. 1993 Apr 15;126(1):109–113. [PubMed]
  • Rose RE. The nucleotide sequence of pACYC184. Nucleic Acids Res. 1988 Jan 11;16(1):355–355. [PMC free article] [PubMed]
  • Schein CH. Solubility as a function of protein structure and solvent components. Biotechnology (N Y) 1990 Apr;8(4):308–317. [PubMed]
  • Sudol M, Greulich H, Newman L, Sarkar A, Sukegawa J, Yamamoto T. A novel Yes-related kinase, Yrk, is expressed at elevated levels in neural and hematopoietic tissues. Oncogene. 1993 Apr;8(4):823–831. [PubMed]
  • Waugh DS, Sauer RT. Single amino acid substitutions uncouple the DNA binding and strand scission activities of Fok I endonuclease. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9596–9600. [PMC free article] [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...