Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Mareike Kurz, Nathan P Cowieson, Gautier Robin, David A Hume, Jennifer L Martin, Bostjan Kobe, Pawel Listwan

Research output: Contribution to journalArticlepeer-review


Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites.
Original languageEnglish
Pages (from-to)68-73
Number of pages6
JournalProtein Expression and Purification
Issue number1
Publication statusPublished - Nov 2006


  • Amino Acid Sequence
  • Animals
  • Antigens
  • Codon
  • Endopeptidases
  • Genetic Vectors
  • Mice
  • Molecular Sequence Data
  • Nuclear Proteins
  • Potyvirus
  • Protein Engineering
  • Proteins
  • Recombinant Proteins
  • Solubility
  • Tobacco
  • Transcription Factors
  • Transcriptional Elongation Factors
  • Upstream Stimulatory Factors


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