Direct Evolution Of Red Fluorescent Biliprotein

Linear tetrapyrrole pigments play a very important role in the absorption of light energy in phycobiliproteins and phytochrome to regulate photosynthesis and photomorphogenesis, especially as the chromophore of fluorescent proteins (FPs). Biliverdin (BV) belongs to tetrapyrrole pigment and is the intermediate in heme catabolism by heme oxygenase1(HO1) in all aerobic organisms. The fluorescence spectrum of many BV-based FPs are near the red zone of the light spectrum, which make them applicable to be the red fluorescent probes.In this study, we expressed the enzyme of HO1in E.coli and constructed a fusion protein named CpeS:HO1, the weak red fluorescence of the two enzyme proteins were innovatively detected by us at622nm and aroused us using directed evolution to enhance the brightness of the two proteins as fluorescent probes.Plasmid pET-30a(+)-cpeS:hol is used as a template to construct mutant library by error-prone PCR. By eight rounds of screening, we find a mutant whose cell color is deeper than the WT after expression. There are two base mutations by sequencing:T388A and T813C, which cause Y130N mutation in its amino acid sequence. Through expression and puficication, we find that the fluorescence quantum yield is slightly decreased from0.015to0.008, while the molar extinction coefficient sharply rised to9895M-1cm-1which is2.51times compared to the WT.Another innovative research is we directly use the enzyme of HO1for random mutation. After screening, a mutant contained A20V in its amino aci sequence are found. The fluorescence quantum yield increased from0.065to0.0734, while the molar extinction coefficient is relatively reduced by9%, both wild type and mutant are not more than5000M-1cm-1, but the cell flurescence of the mutant is2.4times than the wild type, which make it brighter than the WT in fluorescence imaging detection.