Preliminary Crystallographic Study Of A Far-red Fluorescent Protein SmURFP

In 1962 Osamu Shimomura and his team found and isolated the green fluorescent protein from Aequorea victoria jellyfish.Green fluorescent protein and its various mutants are used in biomedical research primarily for labeling and tracing.However,since the emission spectrum of green fluorescent protein is limited to 440-529 nm,the background is high in intracellular imaging,and the labeling and tracer effect applied to living creatures are unsatisfactory.In 1999,red fluorescent protein was first reported,and it's advantage was obvious compared with green fluorescent protein.And the far infrared red fluorescent protein studied in this paper can be a good solution to the problem of high imaging background in vivo.In 2016 Erik A Rodriguez et al.Produced a new far-infrared fluorescent protein named smURFP.The mutant smURFP not only has the highest brightness but also the ability to excite fluorescence in the range of 642-670 nm to meet the requirements for in vivo research.How to understand the structural mechanism of the protein luminescence,and use its biochemical characteristics of transformation,design a new generation of far infrared infrared fluorescent protein has important scientific value and application prospects.In this paper,smURFP was used as a research object,and a series of clones were successfully constructed by molecular cloning,then in E.coli the protein was expressed in large quantities.The smURFP protein with high purity was purified by affinity chromatography,ion exchange chromatography and gel filtration chromatography.The crystallization conditions of smURFP protein were screened to determine the appropriate crystallization buffer.After the pH optimization and additive optimization process,the protein crystals were obtained and the protein was sent to Shanghai Synchrotron radiation for data collection.Although the natural smURFP protein crystallizes to 2?,it is difficult to determine the crystal phase in the data analysis process by molecular replacement method.In order to solve the phase problem,we follow the subsequent smURFP protein selenomethionine labeling,intended to use multi-wavelength anomalous scattering to determine the phase.In the smURFP protein there are four methionine(in addition to the initial methionine),after cloning and purification of the expression of the selenium-labeled protein,we obtained the higher purity selenomethionine-labeled target protein.After the crystallization conditions of the screening and optimize the crystals we get better crystal.Above the preliminary study of smURFP protein will help us to lay the foundation for further analysis of its protein structure,to understand its luminescence mechanism,and to further transform the red fluorescent protein.