Brief Research Report ARTICLE
Single-Input Regulatory Cascade for In Vivo Removal of the Solubility Tag in Fusion Recombinant Proteins Produced by Escherichia coli
- 1Federal University of Bahia, Brazil
Solubility tags are commonly fused to target recombinant proteins to enhance their solubility and stability. In general, these protein tags must be removed to avoid misfolding of the partner protein and to allow for downstream applications. Nevertheless, in vitro tag removal increases process complexity and costs. Herein, we describe a synthetic biology-based strategy to permit in vivo removal of a solubility tag (EDA, KDPG aldolase), through co-expression of the fusion recombinant protein (EDA-EGFP) and the tag-cleaving protease (TEVp), in a controlled manner. Basically, the system uses three repressor proteins (LacI, cI434 and TetR) to regulate the expressions of EDA-EGFP and TEVp, in a regulatory cascade that culminates with release of free soluble target protein (EGFP), following a single chemical induction by IPTG. The system worked consistently when all biological parts were cloned in a single plasmid, pSolubility(SOL)A (7.08 Kb, AmpR), and transformed in Escherichia coli Rosetta (DE3) or BL21(DE3) strains. Total soluble recombinant protein yield (EDA-EGFP + free EGFP) was ca. 272.0 ±60.1 µg/mL of culture, following IMAC purification; free EGFP composed great part (average = 46.5%; maximum = 67.3%) of the total purified protein fraction and was easily separated from remaining fusion EDA-EGFP (53 KDa) through filtration using a 50 KDa cutoff centrifugal filter.
Keywords: Recombinant Proteins, protein solubility, Synthetic Biology, Escherichia coli, green fluorescent protein
Received: 05 Feb 2019;
Accepted: 06 Aug 2019.
Edited by:Pablo Ivan Nikel, The Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain), Denmark
Reviewed by:Michele Galluccio, University of Calabria, Italy
Xristo Zarate, Universidad Autónoma de Nuevo León, Mexico
Copyright: © 2019 Silva, Santos, Meyer, Alcantara-Neves, Pinheiro and Pacheco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Dr. Luis G. Pacheco, Federal University of Bahia, Salvador, 40110-060, Bahia, Brazil, firstname.lastname@example.org