Shapira S K, Casadaban M J
Gene. 1987;52(1):83-94. doi: 10.1016/0378-1119(87)90397-0.
The thymidine kinase (TK) gene (tk) from Herpes simplex virus type 1 has been used to form gene fusions encoding enzymatically active hybrid proteins. The promoter, translation initiation region, and the first three codons of the tk gene were removed and replaced with a series of DNA restriction sites. DNA fragments containing gene initiation regions were cloned into these sites and shown to synthesize enzymatically active proteins in Escherichia coli. These gene fusions were shown to complement an E. coli strain which is deficient in TK function. Gene initiation regions were used from the lac operon, the tnpR gene of Tn3, and the insA gene of ISl. TK synthesis was regulated by the control signals of the promoter fused to tk, and was dependent upon the phase alignment of the codons at the fusion joint. The size of the resulting protein was shown to be increased over the size of the original TK protein by the length of the coding region fused to TK. This demonstrated that the tk gene has non-essential N-terminal amino acids that can be replaced by other amino acid sequences with the retention of TK enzymatic activity. Such tk gene fusions are useful in situations where fusions with other genes cannot be conveniently selected or assayed.
来自单纯疱疹病毒1型的胸苷激酶（TK）基因（tk）已被用于构建编码具有酶活性的杂交蛋白的基因融合体。tk基因的启动子、翻译起始区域和前三个密码子被去除，并用一系列DNA限制性酶切位点取代。含有基因起始区域的DNA片段被克隆到这些位点，并显示在大肠杆菌中合成具有酶活性的蛋白质。这些基因融合体被证明可互补缺乏TK功能的大肠杆菌菌株。使用了来自乳糖操纵子、Tn3的tnpR基因和IS1的insA基因的基因起始区域。TK的合成受与tk融合的启动子的控制信号调节，并取决于融合接头处密码子的相位排列。结果表明，所得蛋白质的大小比原始TK蛋白质的大小增加了与TK融合的编码区域的长度。这表明tk基因具有非必需的N端氨基酸，这些氨基酸可以被其他氨基酸序列取代，同时保留TK酶活性。这种tk基因融合体在与其他基因的融合不能方便地选择或检测的情况下很有用。
