Study Of Expression,Purification And Catclytic Activity Of Self-assembled Fluorinase Aggregates

To our knowledge,Streptomyces xinghaiensis is the first ever fluorinase unveiled from marine source.It has been proved to have a unique ability to utilise inorganic fluorine ions(F-)and S-adenosyl-L-methionine(SAM)to catalyse a reaction to generate 5'-fluorodeoxy adenosine(5'-FDA).And the 5'-FDA can be catalyzed by Trypanosoma vivax nucleoside hydrolase(TvNH)to produce 5'-fluorodeoxyribose(5'-FDR).This significant feature enables fluorinase to incorporate[18F]-fluoride into different small molecules to generate radioactive tracer in PET(positron emission tomography)for medical testing.In addition,fluorinase is greatly valuable in synthetic biology for incorporating fluorine element into building blocks to produce non-natural organofluorines,or as biocatalyst for transforming non-native substrates.In order to obtain fluorinase quickly,efficiently and economically.this topic explored the method of engineering fluorinase at the molecular level.and preliminary research on the properties of novel fluorinase.In the research of this study,the coding sequences of the three self-assembled short peptide tags ELK16,L6KD and 18A had been found to be ligated to the 3'end of the fluorinase gene(JIA)by molecular cloning,respectively.Three expression vectors pET-28a(+)fIA?ELK16,pET-28a(+)fIA-L6KD and pET-28a(+)-fIA-18A were successfully constructed.Expression was induced in E.coli BL21(DE3),and three fluorinase aggregates,namely FIA-ELK16,FIIA-L6KD and FIA-18A,are obtained.At the same time,according to the coding sequence of T.vivax nucleoside hydrolase(TvNH),pET-28a(+)-TvNH expression vector was constructed.And TvNH was successfully obtained after induction.Three fluorinase aggregates were identified by HPLC and LC-MS to catalyze the formation of 5'-FDA.At the same time,through the detection of AFM,TEM and DLS,the three fluorinase aggregates can self-assemble to form protein particles with different nanometer sizes in aqueous solution,FIA-ELK16 had the largest aggregates reaching 500-600 nm in diameter while the aggregates of both FIA-L6KD and FIA-18A were similar in sizes,reaching roughly 200 nm in diameter.The activities of the three fluorinase aggregates correspond to their particle size.In particular,the FIA-L6KD and FIA-18A aggregates had a catalytic efficiency(Keat)of 128%and 180%,respectively,of the soluble fluorinase FIA.Since FIA-ELK16 had the largest particle size,its Keat was 83%of the soluble fluorinase FIA,which also proves that the formation of aggregates does not cause a large loss of enzyme activity.In the thermal stability experiment,it was shown that all three fluorinase aggregates have good thermal stability,especially FIA-L6KD,and their half-life is 1.7 times that of FIA at their optimum temperature;at the same temperature(50?).its half-life is 79 times that of FIA.Repeatability experiments showed that the activity of the three fluorinase aggregates remained above about 50%when used nine times,with FIA-ELK16 and FIA-L6KD remaining above 70%.These two points further illustrate the excellent performance of fluorinase aggregates and have good application potential.Finally,the purified TvNH catalyzed 5'-FDA was used to identify the efficiency of the catalytic fluorinase and fluorinase aggregates to catalyze the formation of 5'-FDR by HPLC and LC-MS analysis.The feasibility of using fluorinase to potentially produce[18F]-fluoride for PET was verified.