Kim J S, Raines R T
Department of Biochemistry, University of Wisconsin, Madison 53706.
J Biol Chem. 1993 Aug 15;268(23):17392-6.
Bovine seminal ribonuclease (BS-RNase), a homolog of bovine pancreatic ribonuclease (RNase A), is isolated as a dimer in which the subunits are cross-linked by two disulfide bonds. In addition to this anomalous quaternary structure, the enzyme has extraordinary biological properties, such as antispermatogenic, antitumor, and immunosuppressive activities. The molecular bases for these properties are well-suited for exploration with the techniques of recombinant DNA. Accordingly, a gene encoding BS-RNase was designed based on criteria expected to maximize the translational efficiency of its mRNA in Escherichia coli. This gene was constructed from 12 synthetic oligonucleotides and expressed with the phage T7 system. The protein thus produced was insoluble and accumulated under optimal conditions to 15% of total cellular protein or 200 mg/liter of culture. Ribonuclease activity was generated by air oxidation of the reduced and denatured protein. Three forms of active BS-RNase were isolated by gel filtration chromatography: the well-characterized dimer and monomer and a previously uncharacterized form that migrated as a trimer. The ribonuclease activities of all three forms were equivalent to or higher than that of dimeric BS-RNase isolated from bull seminal plasma.
牛精核糖核酸酶（BS-RNase）是牛胰核糖核酸酶（RNase A）的同源物，以二聚体形式分离出来，其中亚基通过两个二硫键交联。除了这种异常的四级结构外，该酶还具有非凡的生物学特性，如抗生精、抗肿瘤和免疫抑制活性。这些特性的分子基础非常适合用重组DNA技术进行探索。因此，基于有望使其mRNA在大肠杆菌中翻译效率最大化的标准设计了一个编码BS-RNase的基因。该基因由12个合成寡核苷酸构建而成，并通过噬菌体T7系统进行表达。由此产生的蛋白质不溶，并在最佳条件下积累至总细胞蛋白的15%或每升培养物200毫克。还原和变性的蛋白质通过空气氧化产生核糖核酸酶活性。通过凝胶过滤色谱法分离出三种活性BS-RNase形式：特征明确的二聚体和单体，以及一种以前未鉴定的三聚体形式。所有三种形式的核糖核酸酶活性都等同于或高于从公牛精浆中分离出的二聚体BS-RNase的活性。
