Background: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA.
Methods: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels.
Results: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs.
Conclusions: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.
Keywords: Hexahistidine tag; Isothermal DNA amplification; Recombinase polymerase amplification (RPA); uvsY.
A Strategy to Fight against Triple-Negative Breast Cancer: pH-Responsive Hexahistidine-Metal Assemblies with High-Payload Drugs
Triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer, is difficult to be targeted therapeutically due to negative expression of the bioreceptor, which leads to the poorest overall four-year survival rate among all cancer subtypes.
We proposed that the nanomedicine featuring high payload and pH-responsive release of the loaded drugs could assist the TNBC treatment. In the present study, the His6-metal assemblies (HmA) were employed to encapsulate the doxorubicin (Dox), and the effect of HmA loaded with Dox (HmA@Dox) on treating TNBC was evaluated in vitro and in vivo.
We found that the participation of Dox in the formation of HmA leads to high loading efficiency (99.4% for concentration ≤ 1 mg/mL) and the loading capacity (50.7% for concentration ≥ 10 mg/mL) of Dox encapsulated into HmA. HmA@Dox exhibited a narrow size distribution on the nanoscale, a pH-responsive release of loaded Dox, a quick endocytosis process, and fast lysosome escape. Most importantly, the HmA@Dox showed high efficacy in killing various breast cancer cells (MCF-7, MDA-MB-231, and MDA-MB-453) in vitro and depressing the development of TNBC in vivo.
Our results demonstrated that such a strategy for designing a nanomedicine with high payload and responsive release of drugs to the environment around the tumor was of great importance to treat TNBC.
Efficient Delivery of Antibodies Intracellularly by Co-Assembly with Hexahistidine-Metal Assemblies (HmA)
Purpose: There has been a substantial global market for antibodies, which are based on extracellular targets. Binding intracellular targets by antibodies will bring new chances in antibody therapeutics and a huge market increase. We aim to evaluate the efficiency of a novel delivery system of His6-metal assembly (HmA) in delivering intracellular antibodies and biofunctions of delivered antibodies.
Methods: In this study, the physicochemical properties of HmA@Antibodies generated through co-assembling with antibodies and HmA were well characterized by dynamic light scatter. The cytotoxicity of HmA@Antibodies was investigated by Cell Counting Kit-8 (CCK-8). The endocytic kinetics and lysosome escape process of HmA@Antibodies were studied by flow cytometry and fluorescent staining imaging, respectively. Compared to the commercialized positive control, the intracellular delivery efficiency by HmA@Antibodies and biofunctions of delivered antibodies were evaluated by fluorescent imaging and CCK-8.
Results: Various antibodies (IgG, anti-β-tubulin and anti-NPC) could co-assemble with HmA under a gentle condition, producing nano-sized (~150 nm) and positively charged (~+30 eV) HmA@Antibodies particles with narrow size distribution (PDI ~ 0.15). HmA displayed very low cytotoxicity to divers cells (DCs, HeLa, HCECs, and HRPE) even after 96 h for the feeding concentration ≤100 μg mL-1, and fast escape from endosomes. In the case of delivery IgG, the delivery efficiency into alive cells of HmA was better than a commercial protein delivery reagent (PULSin).
For cases of the anti-β-tubulin and anti-NPC, HmA showed comparable delivery efficiency to their positive controls, but HmA with ability to deliver these antibodies into alive cells was still superior to positive controls delivering antibodies into dead cells through punching holes.
Conclusion: Our results indicate that this strategy is a feasible way to deliver various antibodies intracellularly while preserving their functions, which has great potential in various applications and treating many refractory diseases by intracellular antibody delivery.
Keywords: antibody; coordination polymer; intracellular delivery; nanocarrier; peptide assembly.
Efficient delivery of cytosolic proteins by protein-hexahistidine-metal co-assemblies
Proteins play key roles in most biological processes, and protein dysfunction can cause various diseases. Over the past few decades, tremendous development has occurred in the protein therapeutic market due to the high specificity, low side effects, and low risk of proteins.
Currently, all protein drugs on the market are based on extracellular targeting; more than 70% of intracellular targets remain un-druggable. Efficient delivery of cytosolic proteins is of significance for protein drugs, advanced biotechnology and molecular cell biology. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for intracellular protein delivery.
Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged protein encapsulation particles(Protein@HmA) through a co-assembly process with a high loading capacity and loading efficiency.
Protein@HmA was able to deliver proteins with diverse physicochemical properties through multiple endocytosis pathways, and the protein could quickly escape from endosomes.
In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells. Our results demonstrate that this strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics,
STATEMENT OF SIGNIFICANCE: : Intracellular targets with protein drugs may provide a new way for the treatment of many refractory disease. Herein, we developed a co-assembly strategy for protein-hexahistidine-metal for efficient intracellular protein delivery.
Anti-Hexahistidine (His6) tag Antibody
St John's Laboratory
Recombinant Human MBL2 Protein-Hexahistidine tag
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Based on the coordinative interaction between His6 and metal ions, various proteins were encapsulated in situ into nanosized and positively charged particles (Protein@HmA) with a high loading efficiency.
Protein@HmA was able to deliver different proteins through multiple endocytosis pathways, and the protein could quickly escape from endosomes. In addition, the bioactivity of the loaded protein during co-assembly and the intracellular delivery processes were well preserved and could be properly exerted inside cells.
This strategy should be a valuable platform for protein delivery and has huge potential in protein-based theranostics.