Gawlak S L, Neubauer M, Klei H E, Chang C Y, Einspahr H M, Siegall C B
Molecular Immunology Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Biochemistry. 1997 Mar 18;36(11):3095-103. doi: 10.1021/bi962474+.
Bryonia dioica (Cucurbitaceae family) produces at least two type I ribosome-inactivating proteins, bryodin 1 (BD1) and bryodin 2 (BD2). A cDNA sequence encoding BD1 was isolated from B. dioica leaf mRNA using degenerative oligonucleotides and codes for a 22 amino acid signal peptide followed by a protein of 267 residues. Expression of two recombinant BD1 (rBD1) forms in Escherichia coli yielded proteins of 267 (to the natural stop codon) and 247 amino acids (to the putative cleavage site yielding the mature protein) that had identical protein synthesis inhibition activity as compared to native BD1. The substitution of Lys for Glu at position 189 near the active site reduced the ability of rBD1 to inhibit protein synthesis by 10-fold. Toxicologic analysis showed that rBD1 was well tolerated in rodents with LD50 values of 40 mg/kg in mice and >25 mg/kg in rats. A crystal of mature rBD1 protein was used to collect X-ray diffraction data to 2.1 A resolution. The rBD1 crystal structure was solved and showed extensive homology with other type I RIPs and A chains of type II RIPs. The studies described here demonstrate that rBD1 retains full biologic activity and serve as a guide for using this potent, yet nontoxic, RIP in the construction of single-chain immunotoxin fusion proteins.
雌雄异株泻根（葫芦科）产生至少两种I型核糖体失活蛋白，泻根蛋白1（BD1）和泻根蛋白2（BD2）。利用简并寡核苷酸从雌雄异株泻根叶mRNA中分离出编码BD1的cDNA序列，该序列编码一个22个氨基酸的信号肽，其后是一个267个残基的蛋白质。在大肠杆菌中表达两种重组BD1（rBD1）形式，产生了267个氨基酸（至天然终止密码子）和247个氨基酸（至产生成熟蛋白的推定切割位点）的蛋白质，与天然BD1相比，它们具有相同的蛋白质合成抑制活性。在活性位点附近的第189位将谷氨酸替换为赖氨酸，使rBD1抑制蛋白质合成的能力降低了10倍。毒理学分析表明，rBD1在啮齿动物中耐受性良好，小鼠的半数致死量（LD50）值为40mg/kg，大鼠的LD50值>25mg/kg。利用成熟rBD1蛋白的晶体收集了分辨率为2.1埃的X射线衍射数据。解析了rBD1晶体结构，发现其与其他I型核糖体失活蛋白和II型核糖体失活蛋白的A链具有广泛的同源性。本文所述研究表明，rBD1保留了全部生物活性，并为在构建单链免疫毒素融合蛋白中使用这种强效但无毒的核糖体失活蛋白提供了指导。
