doi:10.1016/j.pep.2006.05.006
Copyright © 2006 Elsevier Inc. All rights reserved.
Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Mareike Kurza, Nathan P. Cowiesona, Gautier Robina, David A. Humea, c, d, Jennifer L. Martina, b, d, Bostjan Kobea, b, d, 
, 
and Pawel Listwana, b, ,
aInstitute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia
bSchool of Molecular and Microbial Sciences, University of Queensland, Brisbane, Qld 4072, Australia
cCooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Qld 4072, Australia
dARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Qld 4072, Australia
Received 31 March 2006;
revised 12 May 2006.
Available online 20 May 2006.
Abstract
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.
Keywords: TEV (tobacco etch virus) protease; Recombinant protein expression in E. coli; Protein purification; Histidine; Protein solubility; E. coli; Fusion protein; Proteolytic cleavage
Fig. 1. Amino acid sequences of the four N-terminal hexahistidine tags. The hexahistidine sequence of each tag is shaded in black, and the TEV protease recognition site (ENLYFQ/G) of the TEV1 and TEV2 tags is shown in bold. For the His-S and TEV2 tags, the encoded product includes an additional four residues (SAND) at the C-terminus.
 |
Fig. 2. Expression and solubility analysis of the selected constructs with four different His-tags. SDS–PAGE analysis of samples of whole cell lysate (W), soluble fraction (S), and elution (E) after IMAC purification (see Materials and methods for details) is shown for two of the test proteins. Boxes indicate the expected mass of the expressed protein. (A) The mouse protein USF1 is expressed in high yield from E. coli when the His-L, His-S, and TEV1 tags are present, but there is no expression observed for the TEV2 tagged protein. In the case of the His-L and His-S tagged proteins, the product is soluble, but for the TEV1 tag the product is a mixture of soluble and insoluble material. (B) Mouse latexin is expressed at high levels with all four tags, but the expressed protein is soluble only when the His-L or His-S tag is present. Incorporation of the TEV recognition site (in the TEV1 and TEV2 tagged proteins) results in insoluble protein. A similar analysis of expression yield (high or low) and solubility (soluble, insoluble, or mixture) was performed on all nine proteins, and the results summarized in Table 1.
Fig. 3. Comparison of ten different vectors containing an N-terminal His-tag and a TEV cleavage site. All vectors contain the N-terminal His-tag, the difference being the length of the linker region, the presence or absence of a leader sequence and the amino acid composition. Our vector (TEV1); pMCSG7 [19]; pET-NH6 [6]; pENTR-His-ccdB [20]; SGC vectors p11, pLIC-SGC1 (http://www.sgc.utoronto.ca/SGC-WebPages/toronto-vectors.php); EMBL Protein Expression and Purification Facility vectors pBADM-11, pETM-20, pETM-50, pProExHTa (http://www.embl-hamburg.de/~geerlof/webPP/vectordb/bact_vectors/table.html).
Table 1.
Expression and solubility of the four constructs of each of the nine mouse proteins
The level of expression (H, high, or L, low) is based on a quantitative assessment of the amount of protein visible in the whole cell extract at the expected molecular mass, relative to the other eight proteins (each of the nine proteins was prepared and analyzed in exactly the same way). Evaluation of the solubility of the expressed protein (S, soluble; I, insoluble; M, mixture of soluble and insoluble product; and −/−, no expression detected) is based on the amount of protein in the soluble and elution fractions relative to the amount of protein in the whole-cell lysate.
— Data not available.
a Riken clone ID: B230118O04.
b Riken clone ID: ZX00037N13.
Corresponding authors. Fax: +61 7 3365 4699.
Abstract
Purchase PDF (440 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (0)
27 min) while the cleavage reaction at the 58/30-kDa junction was judged “fast” (
Purchase PDF (1547 K)
