Boulé J B, Johnson E, Rougeon F, Papanicolaou C
Départment d'Immunologie, Institut Pasteur, Paris, France.
Mol Biotechnol. 1998 Dec;10(3):199-208. doi: 10.1007/BF02740839.
Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.
末端脱氧核苷酸转移酶（TdT）是一种高度保守的脊椎动物酶，具有独特的能力，可催化脱氧核苷5'-三磷酸随机添加到单链DNA的3'-羟基上。它在免疫球蛋白和T细胞受体多样性的产生中起重要作用。TdT通常从动物胸腺中获得或在杆状病毒系统中产生，但这两个过程都相当繁琐，并且在纯化过程中会发生蛋白水解。试图在细菌中过表达TdT的尝试均未成功，或者得到的酶比活性较低。因此，TdT的缺乏阻碍了详细的结构和功能研究。在本研究中，我们报告称，通过降低生长温度并过表达一种罕见的精氨酰tRNA，可以在大肠杆菌中以最小的蛋白水解和高比活性提高鼠TdT的产量。
