Engineering an optimized expression operating unit for improved recombinant protein production in Escherichia coli.

Common strategies to improve recombinant protein production in Escherichia coli often involve the test and optimization of several different variables, when using traditional expression vectors that are commercially available. Now, modern synthetic biology-based strategies allow for extensive modifications of these traditional vectors, or even construction of entirely new modular vectors, so as to permit tunable production of the recombinant proteins of interest. Herein, we describe the engineering of a new expression operating unit (EOU; 938 bp) for producing recombinant proteins in E. coli, through the combinatorial assembly of standardized and well-characterized genetic elements required for transcription and translation (promoter, operator site, RBS, junction RBS-CDS, cloning module, transcriptional terminator). We also constructed a novel T7 promoter variant with increased transcriptional activity (1.7-fold higher), when compared to the canonical wild type T7 promoter sequence. This new EOU yielded an improved production of the reporter protein superfolder GFP (sfGFP) in E. coli BL21(DE3) (relative fluorescence units/RFU = 70.62 ± 1.62 A U.) when compared to a high-producing control expression vector (plasmid BBa_I746909; RFU = 59.68 ± 1.82 A U.). The yields of purified soluble recombinant sfGFP were also higher when using the new EOU (188 mg L-1 culture vs. 108 mg L-1 in the control) and it performed similarly well when inserted into different plasmid backbones (pOPT1.0/AmpR and pOPT2.0/CmR).

Rationally designed hypoallergenic mutant variants of the house dust mite allergen Der p 21.

BACKGROUND: Allergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant. METHODS: Herein, we used a structural bioinformatics and immunoinformatics approach to design hypoallergenic mutant variants of the Der p 21 allergen of Dermatophagoides pteronyssinus, which were then recombinantly expressed in bacteria and tested for their IgE-reactivities. For this, we scanned the wild-type Der p 21 protein for all possible single amino acid substitutions in key IgE-binding regions that could render destabilization of the major epitope regions. RESULTS: Four main substitutions (D82P, K110G, E77G, and E87S) were selected to build mutant variants of the Der p 21 allergen, which were produced in their recombinant forms; two of these variants showed reduced reactivity with IgE. Molecular dynamic simulations and immune simulations demonstrated the overall effects of these mutations on the structural stability of the Der p 21 allergen and on the profile of immune response induced through immunotherapy. CONCLUSIONS: When produced in their recombinant forms, two of the Der p 21 mutant variants, namely proteins K110G and E87S, showed significantly reduced IgE reactivities against sera from HDM-allergic individuals (n = 20; p < 0.001). GENERAL SIGNIFICANCE: This study successfully translated a rational in silico mutagenesis design into low IgE-binding mutant variants of the allergen rDer p 21. These novel hypoallergens are promising to compose next-generation allergen-immunotherapy formulations in near future.

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli.

Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, simplify the recombinant protein purification process, and promote the screening of molecules that fail to remain soluble after tag removal. High yields of soluble target proteins have already been achieved using these protease co-expression systems. Herein, we review approaches for controlled intracellular processing systems tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular processing of recombinant proteins regarding system design features, advantages, and limitations of the various strategies.

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

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.