Site-directed Mutagenesis Of Green Fluorescent Protein And Construction Of Novel T Vectors

The TA cloning technique is one of the simplest and most efficient methods for thecloning of PCR products, and it is especially useful when compatible restriction sites arenot available for sub cloning. DNA fragments from one vector into another. A T-vectorcontains a single T extension at the3terminal that permits efficient ligation of cloningPCR products having3-Aoverhangs. One major disadvantage ofcommon T vectors in theblue-white screening system is the demand of IPTG and X-gal. Another disadvantage is theinstability of recombinant colonies in the T vectors utilizing toxin or cecropin for directscreening positive colonies. Recently, an Enhanced Green fluorescent protein (EGFP) wasused as an indicator for the construction of a cloning T-vector and the recombinants ofinserted DNA fragments and this type of T-vector can be assayed by the color of colonyunder UV irradiation. In this study, a mutant of GFP, Emerald, was used as a cellfluorescence indicator for the construction of sensitive T vectors. The main results are asfollows:1Site-directed mutagenesis: The Emerald gene was site-directed mutated in order to addfour new mutant sites (S72A, N149K, M153T, and I167T) on the basis of EGFP(F64L–S65T–H231L).2In vitro analysis: The expression vector p28SUMO-emerald was constructed and therecombinant protein Emerald was expressed and purified. The ultraviolet/visiblespectroscopy and fluorescence spectrum were identical with those reported in literature.The best columnar crystals were obtained in the39th wells of98-well trays(HamptonScreenⅠ&Ⅱ):2mol/L(NH4)2SO4，0.1mol/LHEPES-NaOH pH7.5,2%PEG400.3In vivo analysis: The gene of Emerald was inserted into vector pUC18. In order toenhance the expression and fluorescence of Emerald, an enhancer was added on theupstream of the emerald gene and the relative vector was pUC18E. The green fluorescenceof recombinants in E.coli strains Dh5α appeared after incubation for18h at37℃withoutany IPTG or X-gal. The fluorescence intensity of soluble total protein triples after theenhancer was introduced.4Synonymous mutation: according to the predicted tertiary structure3D models ofEmerald, two synonymous mutations which were located at the93th and109th amino acidpositions were introduced in the Emerald gene in order to make two restrictionendonucleasecites SnaBⅠand SmaⅠrespectively. The insertion sites of SnaBⅠand SmaⅠ were located at the4thand5th strand ofthe β-barrel respectively.5Construction and calibration of T vectors: two novel simple T-vectors pESN-T andpESM-T vectors were constructed. The PCR products of500bp with a random sequencewere taken as test inserts and were ligated with these two T-vectors, the recombinants werewhite, and the non-recombinants were green under visible light.6Insertion mutation: a trinucleotide (TCC) were introduced in the SnaBⅠ site of pESNand the SmaⅠ site of pESM vectors respectively in order to test the sensitivity of the twoT vectors. The fluorescence of recombinants disappeared and the fluorescence intensity ofsoluble total protein was almost zero. Both of the expression and folding of mutationalEmerald decreased significantly.In conclusion, In vitro and in vivo properties of a green fluorescent protein mutantEmerald were analyzed in E.coli. Two restriction enzymes sites SnaBⅠ and SmaⅠwere chosen as insertion sites to construct T vectors. Two mutation vectors which wereintroduced a trinucleotide (TCC) were constructed, the relative fluorescence ofrecombinants was almost zero, the expression level and folding of mutational Emeralddecreased significantly.