Wilson B S, Kautzer C R, Antelman D E
Hybritech Incorporated, San Diego, CA.
Biotechniques. 1994 Nov;17(5):944-53.
This report describes a method whereby library mutagenesis combined with drug selection was used to generate unique and efficient ribosome-binding sites (RBS) for expressing recombinant proteins in Escherichia coli. The RBS was deleted from a vector expressing beta-lactamase and replaced with a 16-base sequence containing a library of mutations. Selection of the library with ampicillin yielded several unique RBS sequences that were more efficient than ompA RBS for expressing a bacterial (beta-lactamase) and a mammalian protein (single-chain Fv antibody). The described approach provides a practical means to improve recombinant protein expression and, also, provides new sequences to further evaluate the complex regulatory mechanism underlying translation initiation.
本报告描述了一种方法，通过该方法将文库诱变与药物筛选相结合，以生成用于在大肠杆菌中表达重组蛋白的独特且高效的核糖体结合位点（RBS）。从表达β-内酰胺酶的载体中删除RBS，并用包含突变文库的16碱基序列进行替换。用氨苄青霉素对文库进行筛选，得到了几个独特的RBS序列，这些序列在表达细菌蛋白（β-内酰胺酶）和哺乳动物蛋白（单链Fv抗体）方面比ompA RBS更高效。所描述的方法提供了一种提高重组蛋白表达的实用手段，同时也提供了新的序列，以进一步评估翻译起始背后复杂的调控机制。
