The Expression, Characterization And Subcellular Location Of The Synthesized Novel Fuorescent Proteins And Construction And Characterization Of The Mutant

Fluorescent proteins are used intensively in cytology, medicine biology and molecular fields as they remain the property of fluorescence excitable activity after fused with other proteins fluently. However, with the advancement of fluorecent proteins study techonlogies, it demands more fluorescent variants'occurrence. Neverthless, fluorecent proteins obtained traditionally restricted in extracting them directly from orgnism or site-directed mutagenesis and random mutation methods and little attentions payed on gene synthesis technology to get the novel fluorescent proteins.A series of gene fragments which are the same length as the full-length of fluorescent proteins obtained based on the oligonucleotides got by microfluidic array synthesis and in vitra assembling. Then cloned them into pET-28a(+) fusion vector, the combinatorial plasmids of pET-28a(+)-FP were gotten after screening, (we mainly focused on three of them which marked with B11, E6 and H3 for characterization)subsequently transformed them into E.coli BL21(DE3) after identification. Then we optimised their expression according to the factors which affect the proteins expression and lastly gained their optimization expression conditions. Moreover, we got the maturation time of fluorescent proteins by adding protein inhibitor and measured the aliquots'fluorescence intensities which took at different designated time points. Based on fluorescent proteins maturation time and optimization expression condition a amount of proteins were harvested and then after purified with Ni-IDA affinity chromatography which specifically reacted with His tag of FP-his tag and subsequently further purified with FPLC the pure proteins were obtained whose purities were obover 95% and suited for subsequent chractarization. The mass spectra results showed that caculated molecular weights of the three proteins are consisted with thereotical values. and the obsorption spectra data presented that the obsorption peak of B11 and E6 are 484 nm and 547 nm respectively and H3 has two absorption peaks, one located at 385 nm and the other 501 nm, which indicated it is probably that there is an intermediate lasts long during its maturation process; the excitation wavelength and emission wavelength of B11, E6 and H3 are 484 nm, 510 nm; 535 nm, 560 nm and 540 nm, 565 nm respectively, to assume that E6 and H3 are belong to far-red fluorescent proteins. Comparation between B11, EGFP and EBFP for Fluorescence spectra and three-dimensional structure proposed that it is possible the residue of 145 in B11 played an important role in its quantumn yield, in order to testify this, the primers containing mutation site F145Y were designed and after fragement PCR, overlap PCR the flull-length with mutation site was acquired. Last we measured its quantumn yield chang comparation with B11 after indentification, expression and purification and the results apparently showed that the qantumn yield of B11 mutant is 0.24 which is larger than that of the primary B11(0.21), so it ensured our proposition. In order to test whether our protein can express in mammalian cells of Hela, we constructed pcDNA3.1(+)-G2-HexaHis vector and then observed significant fluorescence after transfection into Hela cells. And G2 was located in the plasma of Hela cells after expression with the photo tool of the laser confocal microscope. From above results we can conclude that it is feasible that obtained novle fluroscent proteins with gene synthesis method and our proteins can be used in the studies of proteins location, the transcription intensity measurement and interaction between proteins inditification and they also can be potential tools applied in FRET and muti-labling fluroscent proteins studies.