The Research And Application Of Supercharged Green Fluorescent Protein In Biosensing

Supercharged green fluorescent protein (ScGFP), developed by resurfacing of greenfluorescent protein through genetic modification, is a new class of functional proteinwith high net charge. ScGFP shows exceptional solubility and stability against proteinaggregation, exhibiting great promise for applications in biotechnology, medicine, andeven materials science. Moreover, superpositively charged green proteins are cellpenetrating and are able to efficiently deliver functional nucleic acid and protein intomammalian cell. ScGFPs possess several advantages over conventional deliveryvectors, including low cytotoxicity, high versatility, and generality across various celltypes. The self-assembled polyion complex is formed by superpositive ScGFP andnegative charged nucleic acid via electrostatic interaction, which is similar to thenatural assembly of histone protein with oppositely charged DNA. Due to their robuststability, cell penetrating ability, and strong electrostatic interaction with nucleic acid,the potential of ScGFP as biosensing platform is expected. However, their applicationin bio-detection is scarce. Herein, taking ScGFP (+36) as the recognition element andsignal reporter, we expect to develop a simple and versatile biosensing platform for theanalysis of important biomarkers associated with severe diseases. The main points aresummarized as follows:(1) Construction of prokaryotic expression vector plasmid containing the gene ofScGFP, as well as expression and purification of ScGFP. The gene sequence encodingScGFP (scgfp) was reversely translated from the amino acid sequence of ScGFP andoptimized for E. coli codon usage. The full length gene inserted in plasmid pUC19wasobtained by whole gene synthesis. Plasmid pUC19-scgfp and pET28were mixedtogether in a tube, followed by using “one pot” method to construct recombinantplasmid pET28-scgfp. The “one pot” method developed by us, can accomplish theconstruction of recombinant plasmid in less than2h, without isolation and recovery ofDNA. Then the plasmid pET28-scgfp was transformed into E.coli for the expression ofScGFP. And the purification of ScGFP was performed on an AKTA purifier system byusing Ni-NTA agarose and desalinating columns. ScGFP with high purity wascharacterized by SDS-PAGE.(2) Electrostatic interaction between ScGFP and DNA. Using a20bp dsDNA as themodel, it was found that the ScGFP/DNA nanocomplex was generated really quickly (in20s), with an average diameter of568±46nm, characterized by dynamic lightscattering, and its formation was further demonstrated by atomic force microscopy.Furthermore, the formation of ScGFP/DNA nanocomplex can be directly observed byconfocal fluorescence microscopy. Interestingly, the nanocomplex of ScGFP withDNA caused almost40%fluorescence increase due to the surrounding environmentchange and confined state of ScGFP in nanocomplex. Additionally, the fluorescenceanisotropy of ScGFP decreased significantly from0.3to0.07after being encapsulatedin nanocomplex, which could be reasoned by the homo-FRET among ScGFP moleculesin ScGFP/DNA complex. As the DNA was labeled with a quencher, the fluorescence ofScGFP in the nanocomplex was efficiently quenched with the maximal quenching rateof95%. According to the DNA concentration-dependent quenching curve, thedissociation constant value (Kd) between ScGFP and20bp dsDNA was estimated to be0.33nM.(3) ScGFP as a simple and veritable probe for homogeneous DNA detection and DNAmethylation analysis. As a proof-of-principle, we reported a novel application ofScGFP as versatile biosensing platform for nucleic acid detection and epigeneticsanalysis. Taking ScGFP as the signal reporter, a simple turn-on homogenous methodfor DNA detection has been developed based on the polyion nanocomplex ofScGFP/DNA and toehold strand displacement. This assay shows high sensitivity(detection limit,1nM) and potent ability to detect single-base mismatch (~10times,signal ratio of target to signal base mutation). Furthermore,combined with bisulfiteconversion, this ScGFP-based assay was further applied to analyze site-specific DNAmethylation status of genomic DNA extracted from real human colon carcinoma tissuesample with ultrahigh sensitivity (4amol methylated DNA).(4) ScGFP as a universal biosensing platform for sensitive nuclease andmethyltransferase activity assays. In this work, benefiting from the polyionnanocomplex of ScGFP/DNA, a biosensing platform for sensitive, homogeneous and“switch-on” detection of restriction and nonrestriction endonucleases andmethyltransferases was developed based on ScGFP. The cleavage of DNA probe of byS1nuclease results in the quencher being released from the probe, moving away fromScGFP, and subsequently recovery the fluorescence of ScGFP. Therefore, a facile“turn-on” assay for S1activity detection was achieved with a wide linear range (0.008–0.4U/mL) and a low detection limit of0.002U/mL. Following the same sensingstrategy and a modified DNA probe, this assay was applied to analyze the activity ofrestriction endonuclease EcoRI with high sensitivity (1.3U/mL). Similarly, with the help of methylation-specific restriction endonuclease DpnI, Dam methyltransferaseactivity detection and its inhibitor screening were achieved based on this sensingplaform.(5) H39GFP as a novel sensing platform for label-free detection of metal ion andenzyme activity. Protein resurfacing consists of designing a protein’s surface whilepreserving the overall fold and function of the protein intact. H39GFP, a new kind ofgreen fluorescent protein containing39histidine residues in the primary sequence, wasdeveloped by protein resurfacing recently. H39GFP shows many excellent properties,including exceptional stability, cell penetrating ability and regulatable surface charges.Herein, taking H39GFP as the signal reporter, a label-free fluorometric sensor for Cu2+sensing was developed based on multivalent metal ion binding property o f H39GFP andfluorescence quenching effect of Cu2+. The complexation between Cu2+and H39GFP israpid, with high affinity (Kd=16.2nM). This assay provides a convenient“mix-and-read” approach for Cu2+detection with a linear range from0.05to2.0μMand a detection limit of50nM. Furthermore, this H39GFP-based Cu2+assay was furtherapplied to the detection of acetylcholinesterase (AChE) and its inhibitors screening.AChE hydrolyzes acetylthiocholine (ATC) into thiocholine, which reacts with Cu2+togenerate Cu-(thiocholine)2. The complex restores H39GFP’s fluorescence, therebyresulting in the switch-on detection of AChE avtivity. The sensitive and selectivedetection of AChE was achieved with a detection limit of0.015mU/mL. Tacrine andcarbaryl were employed to inhibit the hydrolysis of ATC. IC50of tacrine was estimatedto be3.5nM, and the inhibition efficiency of carbaryl was found to be linearlydependent on its logarithm concentration with the lowest detectable concentration of7.03×10-9g/L. Furthermore, the detecting results of carbaryl residues in food samplesvia this method agreed well with those from high-performance liquid chromatography.