A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

A Scalable Screening of E. coli Strains for Recombinant Protein Expression

Structural biology projects are highly dependent on the large-scale expression of soluble protein and, for this purpose, heterologous expression using bacteria or yeast as host systems are usually employed. In this scenario, some of the parameters to be optimized include (i) those related to the protein construct, such as the use of a fusion protein, the choice for an N-terminus fusion/tag or a C-terminus fusion/tag; (ii) those related to the expression stage, such as the concentration and selection of inducer agent and temperature expression and (iii) the choice of the host system, which includes the selection of a prokaryotic or eukaryotic cell and the adoption of a strain. The optimization of some of the parameters related to protein expression, stage (ii), is straightforward. On the other hand, the determination of the most suitable parameters related to protein construction requires a new cycle of gene cloning, while the optimization of the host cell is less straightforward. Here, we evaluated a scalable approach for the screening of host cells for protein expression in a structural biology pipeline. We evaluated six Escherichia coli strains looking for the best yield in soluble protein expression using the same strategy for protein construction and gene cloning. For the genes used in this experiment, the Arctic Express (DE3) strain resulted in better yields of soluble proteins. We propose that screening of host cell/strain is feasible, even for smaller laboratories and the experiment as proposed can easily be scalable to a high-throughput approach.

Selection of tobacco etch virus protease variants with enhanced oxidative stability for tag-removal in refolding of two disulfide-rich proteins

Tobacco etch virus protease (TEVp) is a powerful enzymatic reagent for removing fusion tag. In this work, we constructed nine TEVp variants with introducing one to three mutations of C19S, C110S and C130S into the soluble TEVp variant, TEVp5M. Using the C-terminal green fluorescent protein (GFP) variant reporter, all constructs showed different solubility levels among four E. coli strains. The TEVp5M containing the C110S and/or C130S mutations in the hyperoxic strain showed the enhanced the cleavage activity. Addition of dithiothreitol to the cultural medium increased the activity of certain constructs produced in the BL21(DE3), contrary to the added hydrogen peroxide, due to cytoplasmic redox change measured by the redox sensitive GFP construct. The more cysteine residues in the purified TEVp5M were modified specifically than those in the other variants. All purified constructs showed similar specific activities in the presence of 5 mM dithiothreitol. In the buffer containing the compounds to aid disulfide bond formation of the refolded protein, the double mutant TEVp5MC110S/C130S exhibited the highest cleavage efficiency. This variant was efficient for removing the fusion tag after refolding of cellulose-binding module tagged disulfide-rich proteins including bovine enteropeptidase and maize peroxidase absorbed on the regenerated amorphous cellulose.

Truncated Thioredoxin Peptides Serves as an Efficient Fusion Tag for Production of Proinsulin

Background: Insulin is a peptide hormone used for regulating blood glucose levels. Human insulin market is projected to grow at a rate of 12.5% annually. To meet the needs of patients, a cost effective insulin manufacturing strategy has to be developed. This can be achieved by selecting a competent host, ideal fusion tag and streamlined downstream process. Objective: In this article, we have demonstrated that selecting a right fusion partner for expression of toxic proteins like insulin, plays a major role in increasing the recombinant protein yield. Methods: In this article, we have focused on identifying a peptide tag fusion partner for expressing proinsulin by truncating thioredoxin tag. Truncations were carried out from both Amino and Carboxy terminus of the protein and efficiency of truncated sequences was evaluated by expressing it with proinsulin gene. FCTRX (1-15) sequence fused to proinsulin was processed further to establish downstream protocol for purification. Results: Thioredoxin tag was truncated appropriately by considering the fusion tag: protein ratio. A couple of sequences ranging 10 – 15 amino acids were identified based on its in silico properties. Of these FCTRX (1-15) showed increased expression and stability of fusion protein. 156 mg of purified insulin was generated from 1g of inclusion body after enzymatic conversion and chromatographic steps. Conclusion: As a result of the current study, it was concluded that FCTRX (1-15) peptide has advantageous attributes to be considered as an ideal fusion tag for expression of proinsulin. This can be further explored by expressing it with other proteins.