Construction Of Protein Delivery Vehicle And Biosensing Platform Based On Protein Engineering

Proteins play an indispensable role in living organisms.Various proteins with different functions make the diversity and complexity of creatures,and present broad applications in scientific research.Further,through genetic engineering and protein engineering,the functions of natural proteins can be modified,as well as combination of different proteins with different functions.Protein therapeutics,with advantages of higher specificity c ompared to small molecular drugs,and lower risk for potential genetic adverse effects than gene therapy,play important roles in biomedicine.However,the cytomembrane of eukaryotic cells is impermeable to most of exogenous proteins.Hence,promising carr iers that integrate high transmembrane ability,improved selectivity,efficient endosome escape,and label-free tracing are in highly demand.The extracellular pH of normal cells is neutral,while the extracellular pH of some lesion cells,such as tumors,are slightly acidic.Designing a carrier that can accurately distinguish the target acidic environment from the normal physiological neutral environment has the potential to improve the selectivity of protein drugs and achieve better therapeutic results.Click chemistry is a class of reactions that mimic the attachment of modular units in nature.Due to its high efficiency and selectivity,and modular blocks,click chemistry has found broad applications in pharmaceutical science,materials chemistry and biological chemistry.There are a large number of site-specific ligation reactions between biomolecules in nature.Discovering click-like reactions with high-reaction rates and specificity from these sources is important for biosensing and material assembly.In this paper,we have constructed the following protein delivery system and biosensing platform based on protein engineering:?1?Design of charge-tunable GFP with precise target pH response properties.superpositively charged green fluorescent proteins?ScGFP?has high cellular uptake potency and stable fluorescence,and can tolerate a certain degree of modification.Based on the charge-dependent transmembrane efficiency of ScGFP and the pH-dependent protonation degree of the imidazole side chain of His,we envisioned that by tuning ratio of His to Lys/Arg,His containing GFP?HisGFP?accurately responding to target pH may be designed as pH sensitive protein carrier.To test this hypothesis,the extracellular pH of most solid tumors,pH 6.5,was chosen as a model target pH,and His₂₉GFP as well as His₁₉GFP were designed.His₂₉GFP can distinguish the small pH difference between tumor extracellular acidity?pH 6.5?and normal physiological pH?pH 7.4?,with high cell uptake potency at pH 6.5,and very low cell uptake potency at pH 7.4.In addition,a large amount of His endows His₂₉GFP with strong proton sponge effect and endosome escape ability.The pH-responsive transmembrane ability and superior endosome escape performance make His ₂₉GFP great potential in protein delivery.?2?Target pH-responsive GFP for intracellular delivery and tracing of functional proteins.Using ribonuclease A?RNase A?and monomeric infrared fluorescent protein?mIFP?as functional protein models,an intracellular functional protein delivery and tracing system with target pH response property was constructed based on His ₂₉GFP.His₂₉GFP successfully delivered these two functional proteins into cells at pH 6.5.RNase A delivered by His₂₉GFP degraded a large amount of intracellular RNA and further led to cell death.Therefore,the delivery system based on HisGFP successfully delivers bioactive proteins into cells under target pH environment.Furthermore,the fluorescence of GFP enables it to track non-fluorescent cargo proteins effectively.These characteristics make it possible for HisGFP with target pH sensitivity to become a universal functional protein carrier in clinical and imaging fields.?3?Mn²⁺-activated DCV-DNA conjunction?MnDDC?.The site-specific protein-DNA interaction is of great importance in the precise regulation of physiological activities,materials science and analytical sensing.The covalent conjunction between duck circovirus replication initiation protein?DCV?and its recognized DNA plays a key role during viral replication.DCV protein was obtained by cloning and expression,and the covalent linkage reaction between DCV and DNA was studied.The reactionperformed in aqueous environment with 90%connection efficiency within two minutes.In addition,the reaction was robust under fluctuating conditions?temperature,pH and buffer?and can still be carried out at low temperature?4??.Moreover,DCV showed excellent selectivity toward Mn²⁺over other divalent metal ions,such as Ca²⁺and Mg²⁺.Therefore,Mn²⁺-activated DCV-DNA conjunction?MnDDC?has obvious click reaction characteristics.Based on the above characteristics,the MnDDC reaction has a good application potential in biosensing or biomolecular assemblies.?4?Construction of Mn²⁺sensor based on the Mn²⁺-activated DCV-DNA conjunction?MnDDC?.Mn²⁺is an essential trace element in human body.Previous studies revealed that Mn²⁺deficiency can result in skeletal defects and abnormal glucose tolerance,while Mn²⁺over exposure are closely related to various diseases,especially neurodegenerative diseases.It is difficult to detect or image Mn²⁺in complex samples,as the paramagnetism of Mn²⁺leads to fluorescence quenching,and the Mn²⁺structure is similar to Ca²⁺/Mg²⁺.On the basis of MnDCC,a fluorescent Mn²⁺biosensor composed of DCV and a molecular beacon,was developed.The sensor exhibited high selectivity and sensitivity towards Mn²⁺with a detection limit of 50 nM,and a linear range of 0.05-8?M and 8-800?M.Using the proposed biosensor,not only analysis of Mn²⁺in real samples?e.g.serum and food?,but also wash-free fluorescent imaging of Mn²⁺in extracellular environment and cytoplasm have been achieved.Moreover,taking human neuroblastoma cell as an in vitro model,for the first time,increase of Mn²⁺concentration in cytosol or in the microenvironment of the cells,generation of oxygen species?ROS?,and down regulation of tyrosine hydroxylase?TH?in the same cell sample have been measured through imaging,thus providing a new method for study of the Mn²⁺-related neurodegenerative diseases.Accordingly,our work demonstrated that the click-type MnDDC is suitable for site-specific covalent protein-DNA linkage in complex biological environments,which sheds light on potential applications of natural click type reactions for bioanalysis and biological research.