大腸桿菌Thioredoxin表現量不同與質體f1 複製原點對噬菌體感染之影響

Abstract

從古細菌至人類的所有物種的細胞中皆存在的thioredoxin，是一個具有熱穩定性的蛋白質，分子量一般約為12 kDa。在大腸桿菌中除了可以參與去氧核糖核酸的形成，並且在噬菌體T7感染時能作為T7 DNA聚合酵素(DNA polymerase)的次單元，幫助聚合酵素與單股DNA結合；在線形噬菌體感染時可以與pI 蛋白結合，幫助脫去pV蛋白，為噬菌體組合的必要成份。 本實驗中利用定點突變改變大腸桿菌thioredoxin活性區裡第33,34位置的氨基酸，並將基因置於不同載體中表現。突變株P34Y與G33V/P34Y分別會讓噬菌體T7/T3的感染平盤效率（E.O.P.）值下降109倍與77倍；這兩個突變株也分別在線形噬菌體的E.O.P值下降1.2x105倍與140倍。突變株P34G只能讓T7/T3的E.O.P.值下降43倍，但是對線形噬菌體的感染能力無明顯影響。除此之外，大量表現突變株thioredoxin時也會影響上述結果，讓噬菌體的感染能力恢復到與野生株類似。 根據本實驗室過去的實驗中發現pET32c上所攜帶的f1 複製原點可能會使線形噬菌體的感染能力下降。因此將基因接至與pET32c類似但不攜帶f1 origin的pET12a，發現以pET12a為載體表現thioredoxin時，被噬菌體f1所感染的E.O.P.會較pET32高19倍；被M13噬菌體所感染時會高3倍。

Thioredoxin is a 12-kDa-heat-stable protein, which present in all living orgnisms ranging from archaebacteria to humans. Escherichia coli thioredoxin is required for the DNA replication of bacteriophage T7 and the assembly of the filamentous phage. We use site-directed mutagnesis to construct three mutants of E.coli thioredoxin to understand the effects of the different amino acid substitutions. The mutants were characterized using an in vivo assay based on the ability of cell to support growth of T7 and filamentous phage. In ordor to express mutant thioredoxin, these mutated trxA genes were constructed in different vectors. P34Y and G33V/P34Y decrease the efficient of plating (E.O.P.) of both kinds of phage. The E.O.P. decreased by 109 and 77 folds for P34Y and G33V/P34Y respectively infected by phage T7/T3. and 1.2x10 9 and 140 folds for P34Y and G33V/P34Y infected by filamentous phage. P34G only decreases the infection ability of T7 slightly. High level expression of the mutated thioredoxin increased infection ability of phages. Wild-type trxA gene was inserted into pET12a, a plasmid does not carry f1 origin. The results indicate that the f1 origin of plasmids can decrease the infection ability of phages. The changes of E.O.P. are 19 and 3 folds in the infection of phage f1 and M13.