An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins

There is a high demand for the production of recombinant proteins in Escherichia coli for biotechnological applications but their production is still limited by their insolubility. Fusion tags have been successfully used to enhance the solubility of aggregation-prone proteins; however, smaller and more powerful tags are desired for increasing the yield and quality of target proteins. Herein, NEXT tag, a 53 amino acid-length solubility enhancer, is described. The NEXT tag showed outstanding ability to improve both in vivo and in vitro solubilities with minimal effect on passenger proteins. The C-terminal region of the tag was mostly responsible for in vitro solubility, while the N-terminal region was essential for in vivo soluble expression. The NEXT tag appeared to be intrinsically disordered and seemed to exclude neighboring molecules and prevent protein aggregation by acting as an entropic bristle. This novel peptide tag should have general use as a fusion partner to increase the yield and quality of difficult-to-express proteins.

Exercise-induced peptide TAG-23 protects cardiomyocytes from reperfusion injury through regulating PKG–cCbl interaction

Recent studies have revealed that proper exercise can reduce the risk of chronic disease and is beneficial to the body. Peptides have been shown to play an important role in various pathological processes, including cardiovascular diseases. However, little is known about the role of exercise-induced peptides in cardiovascular disease. We aimed to explore the function and mechanism of TAG-23 peptide in reperfusion injury and oxidative stress. Treatment with TAG-23 peptide significantly improved cell viability, the mitochondrial membrane potential, and ROS levels and reduced LDH release, the apoptosis rate and caspase 3 activation in vitro. In vivo, TAG-23 ameliorated MI and heart failure induced by I/R or DOX treatment. Pull-down assays showed that TAG-23 can bind to PKG . The TAG-23-PKG complex inhibited PKG degradation through the UPS. We also identified cCbl as the E3 ligase of PKG and found that the interaction between these proteins was impaired by TAG-23 treatment. In addition, we provided evidence that TAG-23 mediated Lys48-linked polyubiquitination and subsequent proteasomal degradation. Our results reveal that a novel exercise-induced peptide, TAG-23, can inhibit PKG degradation by serving as a competitive binding peptide to attenuate the formation of the PKG–cCbl complex. Treatment with TAG-23 may be a new therapeutic approach for reperfusion injury.

A guide-tag system controlling client enrichment into Y15 peptide-based granules for an in-cell protein recruitment assay

A series of guide-tags that can control the enrichment of client proteins into artificial scaffolds constituted by the self-assembling Y15 peptide tag facilitates the analysis of protein–protein interactions in living cells.

Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag

In studies of HIV-1, virus production is normally monitored by either a reverse transcriptase assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. For example, sample dilution is always required in the ELISA procedure to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. This peptide is a fragment of NanoLuc luciferase and generates a strong luminescent signal when complemented with the remaining subunit. To employ this technology, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the resultant virus production, infectivity, or susceptibility to an integrase inhibitor. EM revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1/lentivirus production. This system can be widely applied to a variety of virological studies, along with screening for candidates of future antiviral drugs.