![]() ![]() However, these post-processing steps increase production costs and process intricacy ( Li, 2011). For this, specific protease cleavage sites are placed in between the fusion tag and the target protein, which can then 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. Following expression of the fused recombinant protein, these protein tags need to be detached as they can significantly affect a given passenger protein's biological function. Commonly used solubility enhancers include Maltose-binding protein (MBP, 42.5 KDa), Glutathione-S-transferase (GST, 26 KDa), Thioredoxin A (TrxA, 12 KDa), and N-utilization substance protein A (NusA, 55 KDa). A fusion tag can enhance a given recombinant protein quality by improving its translation, avoiding protein aggregation and even shielding it from degradation ( Waugh, 2005 Kang et al., 2015 Bernier et al., 2018). 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.įusion protein tags are normally used for successfully obtaining hard-to-express recombinant proteins in their soluble form in bacteria. Total soluble recombinant protein yield (EDA-EGFP + free EGFP) was ca. The system worked consistently when all biological parts were cloned in a single plasmid, pSolubility(SOL)A (7.08 Kb, Amp R), and transformed in Escherichia coli Rosetta (DE3) or BL21(DE3) strains. 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. 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. Nevertheless, in vitro tag removal increases process complexity and costs. ![]() In general, these protein tags must be removed to avoid misfolding of the partner protein and to allow for downstream applications. ![]() Solubility tags are commonly fused to target recombinant proteins to enhance their solubility and stability. Post-graduate Program in Biotechnology, Institute of Health Sciences, Federal University of Bahia, Salvador, Brazil. ![]()
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