Single-Input Regulatory Cascade for in vivo Removal of the Solubility Tag in Fusion Recombinant Proteins Produced by Escherichia coli.

Filipe S R Silva,Carina S Pinheiro,Neuza M Alcantara-Neves,Luis G C Pacheco,Roberto Meyer,Sara P O Santos

doi:10.3389/fbioe.2019.00200

Filipe S R Silva, Carina S Pinheiro + Show 4 more

Open Access

https://doi.org/10.3389/fbioe.2019.00200

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Abstract

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 the 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 cut-off centrifugal filter.

Highlights

Fusion protein tags are normally used for successfully obtaining hard-to-express recombinant proteins in their soluble form in bacteria
Specific protease cleavage sites are placed in between the fusion tag and the target protein, which can be recovered in its natural form after in vitro incubation with the respective proteases, such as the Tobacco Etch Virus protease (TEVp), followed by chromatographic steps
The expected functioning of the system is the following: upon Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, the target fusion protein (EDA-enhanced green fluorescent protein (EGFP)) is produced along with the phage 434 repressor protein (cI434) repressor; cI434 in turn binds to its cognate operator site and stops TetR production; TEV protease, which is repressed by binding of TetR to tetO operator site, starts to be produced (Figure 1B; Supplementary Figure S1A)

Summary

Introduction

Fusion protein tags are normally used for successfully obtaining hard-to-express recombinant proteins in their soluble form in bacteria. To the repressilator genetic circuit (Elowitz and Leibler, 2000), our system uses three repressor proteins (LacI, cI434, and TetR) to regulate the expression of the target fusion protein and the TEVp, in a regulatory cascade that culminates with in vivo release of EGFP from its solubility tag (Figures 1A,B).

Results

Conclusion