Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins.
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.
Kurz
Mareike
M
Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia.
Cowieson
Nathan P
NP
Robin
Gautier
G
Hume
David A
DA
Martin
Jennifer L
JL
Kobe
Bostjan
B
Listwan
Pawel
P
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000067699:Ataxin-3; D003062:Codon / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry*; D005822:Genetic Vectors / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017800:Potyvirus / Q000235:genetics; D015202:Protein Engineering; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D014026:Nicotiana / Q000821:virology; D035602:Transcriptional Elongation Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051720:Upstream Stimulatory Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D000941:Antigens; D003062:Codon; D047908:Intracellular Signaling Peptides and Proteins; C086812:Lxn protein, rat; C516959:Memo1 protein, mouse; D009687:Nuclear Proteins; D011506:Proteins; D011994:Recombinant Proteins; C516960:Serf2 protein, mouse; D035602:Transcriptional Elongation Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C534421:OXSR1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D010450:Endopeptidases; C099488:TEV protease; D000067699:Ataxin-3; C511594:Atxn3 protein, mouse