Reconstruction Of The Fluorescent Protein ILOV2Prokaryotic Expression And Crystal Screening

Fluorescent protein derived from light, oxygen and voltage (LOV) domainsoffers many advantages over the green fluorescent protein (GFP) from a smallmolecular weight and efficacy under anaerobic condition. Fluorescent proteins (FPs)have revolutionized the imaging of dynamic processes within living cells (1). Despitetheir wide range of applications, however, green fluorescent protein (GFP) and itsderivatives have three key limitations. First, their use is restricted to aerobic systemsas formation of the chromophore is oxygen-dependent. Second, for certainapplications, the relatively large size of GFP is limiting. Generation of smaller GFPderivatives is unlikely as the11-stranded-barrel structure of the protein is intrinsic toits function. Third, GFP fluorescence is unstable at low pH, prompting thedevelopment of pH-resistant variants (1). As a result of these factors, there isconsiderable interest in developing alternative protein based fluorescent probes.iLOV2fluorescent protein embedded with dichloro-tyrosine possess newfeatures-efficacy under low pH conditions, GFP do not have the major advantages.Second, development of a small molecular weight, non-dependent oxygen, lowpH-resistant fluorescent probes can become a reality. The introduction of non-naturalamino acids to iLOV2fluorescent protein changes the structure of the protein and itsfunction. It is the first time in reconstruction of iLOV2, so it has a lot of innovation.The other experimental technique of the paper is gene codon extensiontechnology. The specific site of the protein is mutated to TAG stop codon and thehighly specific chlorinated tyrosine aminoacyl-tRNA synthase screened can binddichloro-tyrosine tRNA and dichloro-tyrosine The codon extension experimentaltechniques can introduce non-natural amino acid to the protein. The conventionalchemical synthesis method has many drawbacks, for example, low yields, lots ofbyproducts and toxicity. Codon expansion techniques used in protein expression is:mutant plasmid carrying the TAG and PEVOL plasmid including gene of aminoacyltRNA synthetase, the tRNA were transformed to BL21expression strains together. TAG termination codon releasing factor also exists in BL21expression bacteria, soTAG sites of mRNA will appear competitive binding. SDS-PAGE electrophoresis ofinduced whole bacteria can see two strips including two rich proteins, the protein ofsmaller molecular weight is no meaning protein because of protein translationtermination at TAG site, while the other is that dichloro tyrosine is inserted into thefull-length protein successfully and it is a functional protein.Non-natural amino acid aminoacyl-tRNA synthase has a defect in the GSTfusion tag expression because protein insertion translation efficiency of non-naturalamino acids is very low and the specific reason is unclear. Therefore, histidine-taggedwild-type and insert dichloro-tyrosine protein crystals grown successfully. iLOV2protein embedded dichloro-tyrosine has changed the biological function of the proteindramatically, so our results laid the foundation for protein Cl2Y-ilov2structureanalysis.