Research On The Biomolecular Fluorescence Complementation System Based On MNeonGreen And MNeptune

It's of crucial importance to understand the processes of life by studying protein-protein interactions in living cells.In recent years,bimolecular fluorescence complementation(BiFC)has been widely used in detection of protein-protein interaction and its location in vivo.While comparing with other technologies being used to study the in vivo protein interaction,such as yeast two-hybrid,protein fragments complementation,bioluminescence resonance energy transfer,fluorescence resonance energy transfer,and so on,BiFC has the characteristics of high sensitivity,low noise,directness and ease for operation.But the slow maturation of the fluorescent proteins' chromophore limits its applications to study visualization of transient or dynamic interactions in cells.At the same time,there are only a few fluorescent proteins(Cerulean,Citrine,Venus,etc.)that have been used in BiFC and fluorescence complementation can occur under physiological temperature,so we need to develop new BiFC system urgently.Based on the mNeonGreen protein from Branchiostoma lanceolatum,this study developed a BiFC system which has the highest brightness and the shortest maturation time.mNeonGreen is a monomeric yellow green fluorescent protein which is modified from tetrameric LanYFP,its brightness is 1.5 and 3 times as bright as most commonly used GFPs and YFPs,respectively and its oxygen-dependent maturation time(less than 10 minutes)under physiological conditions is the shortest of the fluorescent proteins that have been reported so far.In this study,we obtained the humanized mNeonGreen gene by codon optimization of mNeonGreen gene sequence.And then split the mNeonGreen respectively between three loci(Alal34-Asp135?158Asp-159Lys?Asn173-Gly174)based on its crystal structure and developed a new BiFC system based on mNeonGreen by using a pair of interacting parallel leucine zipper(bFos and bJun)screened that the two fragments when split between Asn173?Gly174 can produce strong fluorescent signal(the other two split ways have no fluorescent signal)in HEK 293T cells.The system has shortened the response time of BiFC system and improved the sensitivity of BiFC system,which is beneficial to the application of BiFC technology in the study of visualization of transient or dynamic interaction in living cells.The second part of this research work was on the problem of the near infrared(NIR)optical window(650-900 nm)of cell imaging in mammalian cells,in-depth studying and optimizing the red BiFC system which has long wavelength(600/651nm)and short maturation time(T1/2?28min)at 37 ?.This study was based on the mNeptune 1 protein BiFC system which has long wavelength(650-900 nm),split the mNeptune 2(599/651 nm)fluorescent protein at the same site(155-156),found that the fluorescence intensity is much weaker than the mNeptunel BiFC system.So,we recombined the different N-peptide segments and C-peptide segments and found that the combination of bJun-Nl+bFos-C2 was the brightest and the brightness is one time more than the mNeptune 1 BiFC system(bJun-Nl+bFos-Cl).In order to explore the mechanism of fluorescence brightness enhancement,we purified the bJun-N1+bFos-C1 and bJun-N1+bFos-C2 proteins respectively in vitro,by detecting their circular dichromatic spectra found that there was no significant difference in the secondary structure between these two BiFC systems,the mechanism of fluorescence intensity enhancement of the new BiFC system is still to be further explored.In addition,this research also use the above two new BiFC systems,to study the fusion ways of the split fluorescent proteins and the interacting proteins(leucine zipper).The results showed that the fusion method of bJun-N+bFos-C was better than the fusion method of N-bJun+bFos-C and bJun-N + C-bFos.This research provides guidance for the wide application of the pair of bFos and bJun BiFC system.