Date Published: February 1, 2019
Publisher: Public Library of Science
Author(s): Ricardo A. Orbegozo-Medina, Victoria Martínez-Sernández, María J. Perteguer, Ana Hernández-González, Mercedes Mezo, Marta González-Warleta, Fernanda Romarís, Esperanza Paniagua, Teresa Gárate, Florencio M. Ubeira, Vikas Nanda.
Recombinant proteins expressed in E. coli are frequently purified by immobilized metal affinity chromatography (IMAC). By means of this technique, tagged proteins containing a polyhistidine sequence can be obtained up to 95% pure in a single step, but some host proteins also bind with great affinity to metal ions and contaminate the sample. A way to overcome this problem is to include a second tag that is recognized by a preexistent monoclonal antibody (mAb) in the gene encoding the target protein, allowing further purification. With this strategy, the recombinant protein can be directly used as target in capture ELISA using plates sensitized with the corresponding mAb. As a proof of concept, in this study we engineered a Trichinella-derived tag (MTFSVPIS, recognized by mAb US9) into a His-tagged recombinant Fasciola antigen (rFhLAP) to make a new chimeric recombinant protein (rUS9-FhLAP), and tested its specificity in capture and indirect ELISAs with sera from sheep and cattle. FhLAP was selected since it was previously reported to be immunogenic in ruminants and is expressed in soluble form in E. coli, which anticipates a higher contamination by host proteins than proteins expressed in inclusion bodies. Our results showed that a large number of sera from non-infected ruminants (mainly cattle) reacted in indirect ELISA with rUS9-FhLAP after single-step purification by IMAC, but that this reactivity disappeared testing the same antigen in capture ELISA with mAb US9. These results demonstrate that the 6XHis and US9 tags can be combined when double purification of recombinant proteins is required.
The detection of serum antibodies induced by parasitic infections using ELISA methods depends on the availability of enough amounts of specific antigens, either natural or recombinant, to be used in the immunoassays. For this purpose, it would be expected that recombinant antigens were more specific than natural ones, but several studies have shown just the contrary [1–4]. Although this phenomenon has not yet been investigated in detail, some reports pointed out that the presence of contaminants derived from the expression of His-tagged proteins in E. coli may explain the poor specificity of such antigens . Optimized E. coli expression , acidic washes [7,8], and washing with low amounts of imidazole  are methods commonly used to prevent the co-purification of proteins on immobilized metal affinity chromatography (IMAC), thus improving the purity of His-tagged recombinant proteins. Also, disulfide bond formation between the protein of interest and other proteins, as well as nonspecific hydrophobic interactions, can be minimized by inclusion of 2-mercaptoethanol and non-ionic detergents, respectively, in the loading buffer . However, since a relevant fraction of contaminant E. coli proteins exhibit moderate to strong affinity for metal-chelating resins , these methods do not guarantee complete purity of recombinant proteins and may decrease the yield of the purified protein.
Epitope tagging is a genetic engineering technique in which a protein encoded by a cloned gene is fused to an epitope recognized by a known antibody [37,38]. This strategy allows rapid affinity-purification of recombinant proteins, but it can also be used for protein detection in WB, flow cytometry and ELISA . Typically, epitope tags (commonly, 5–16 residues in length) are selected among linear sequences [10,40] and have a negative GRAVY index (i.e., hydrophilic) assuming that hydrophilic tags are exposed at the surface and thus are more accessible to antibodies. Also, in most studies tags have been placed at the amino or carboxy terminal regions in the belief that the loss of function will be minimal, although this is not a general rule applicable to any protein (reviewed in ).