Optimization And Application Of High-Performance Beidou Fluorescent Protein

An important component of the phycobilisomes is phycobiliprotein,and the apoprotein of phycobiliproteins can bind different phycobilins,which resulting in phycobiliproteins with different spectral properties.With the advantages of high penetration,low toxicity to cells,low tissue damage and high fluorescence brightness,nearinfrared fluorescent probes have a wide application prospect in the field of in vivo imaging.At present,the available fluorescent probes do not meet all the needs of biological research,and more high-quality fluorescent proteins are needed to cope with the increasing research demands.The spectral properties of phycobiliprotein make it more suitable for studying in vivo near-infrared fluorescent probes.In order to obtain a fluorescent protein with excellent properties,we used the allophycocyanin gene as a template to perform random mutagenesis experiments and introduced the mutated gene into a plasmid containing the T7 promoter induction system.The cells were then induced with protein expression in the model organism E.coli,resulting in the screening of mutants with excellent fluorescence quantum yields,excellent molar extinction coefficients and significantly enhanced effective fluorescence brightness.In order to obtain a fluorescent protein with better performance,this experiment was based on ΔBDFP1.9,a mutant library was constructed by random mutagenesis experiment,and the mutants screened by random mutagenesis were recombined with BV chromophore for heterologous expression.The first is to carry out preliminary screening by measuring the cellular fluorescence spectrum of mutants,then protein purification was performed to purify the proteins of the mutants,and relevant parameters were calculated based on the protein absorption and fluorescence spectra of the mutants.The selected mutants will be sequenced to determine the specific mutation sites,and finally comprehensively screened according to the advantages of each mutant.Eleven mutants were selected by recombinant heterologous expression,and the absorption peak of each mutant protein was around 660 nm and the fluorescence peak was around 670 nm after binding to BV chromophore.The protein fluorescence of most mutants was increased to a certain extent compared with the template ΔBDFP1.9,and the protein fluorescence of Variant-9 increased the most,and its fluorescence peak increased by 3.2times.Compared to template ΔBDFP1.9,the absorption and fluorescence of Variant-2 and Variant-5 were both redshifted to some extent,with the absorption peak of Variant-2redshifted by 4 nm and that of Variant-5 redshifted by 7 nm,and the molecular brightness was increased by 1.43 times and 1.5 times respectively.The molecular brightness of all mutants was enhanced,with Variant-8 having the most significant improvement of 2.49times;Variant-1 having a relatively small increase in molecular brightness,only 1.26 times higher than the template.The expression results of transfected animal cells showed that the mutants and templates were successfully expressed in animal cells,and the relative effective brightness of Variant-1 was increased by about 24%,the relative effective brightness of Variant-2 was increased by about 30%,the relative effective brightness of Variant-4 was increased by about 17%,the relative effective brightness of Variant-5 was increased by about 35%,and the relative effective brightness of Variant-8 was increased by about 8%.Finally,the stability of Variant-1,2,4,5,and 8 with different gradients of pH,different concentrations of guanidine hydrochloride,and time gradients at constant temperature was investigated in cells where the effective fluorescence brightness was enhanced.The pH tolerance of Variant-1,2,4,5,8 and template is basically the same,and they are relatively more suitable for acidic labelling environments.Low concentrations of guanidine hydrochloride have relatively little effect on template and mutant protein fluorescence,but high concentrations of guanidine hydrochloride will destroy protein fluorescence;Template and mutant proteins can adapt to short-term high temperature environments,but long high temperature environments will affect the mutant protein fluorescence signal.