Background: In the process of recombinant protein biosynthesis affinity tags are efficient
tools to achieve the expected purity and yield during the purification steps. Nonetheless
these tags might alter enzyme specificity and activity, therefore in functional assays
it is recommended to use authentic or native proteins. Several ubiquitin fusion systems
have been developed for E. coli-based recombinant protein expression that provide
high levels of expression, with simple purification, and allow the production of various
proteins with authentic N-terminus for subsequent applications. Results: In the present
research, we describe an ubiquitin fused bacterial biosynthetic system (pUbiq) for
the production of the native Second mitochondria-derived activator of caspases (SMAC)
recombinant protein. Using this system, the recombinant protein is expressed with
an ubiquitin-decahistidine fusion partner, then purified from the cell-forming proteins
by affinity chromatography. The fusion partner is then removed by proteolytic digestion,
resulting the native structure of the recombinant protein without unnecessary amino
acid residues. Following proteolysis, another affinity chromatography method is used
to separate the native protein from the fusion partner and the proteolytic enzyme.
The folding of the protein of interest was verified by a pull-down assay. Conclusions:
Based on our results, the presented pUbiq system was successfully applied in the production
of native SMAC recombinant protein, where the affinity tag required for purification
was completely removed. Our study suggests that the ubiquitin-fusion technology will
be useful for enhancing expression and purification of native and authentic proteins
for structural and functional studies as well as for therapeutic uses. How to cite:
Salamona P, Orbana CK, Molnar-Nagy K, et al. Exon based amplified polymorphism (EBAP):
A novel and universal molecular marker for plants. Electron J Biotechnol 2022;56.
https://doi.org/10.10 16/j.ejbt.2022.01.002. (c) 2022 Universidad Catolica de Valparaiso.
Production and hosting by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).