Springer B A, Sligar S G
Department of Biochemistry, University of Illinois, Urbana 61801.
Proc Natl Acad Sci U S A. 1987 Dec;84(24):8961-5. doi: 10.1073/pnas.84.24.8961.
Sperm whale myoglobin was expressed in Escherichia coli from a totally synthetic gene inserted in the expression vector pUC19. The gene was constructed as 23 overlapping oligonucleotides encoding both strands of the DNA. Gene synthesis provides several advantages over traditional eukaryotic gene-cloning techniques, allowing the incorporation of an efficient ribosome binding site, appropriate initiation and termination sequences, restriction enzyme sites for convenient subcloning and future mutagenesis, and frequently used codons for highly expressed E. coli genes. The sperm whale myoglobin expressed from the synthetic gene constituted approximately 10% of the total soluble protein as holo-protein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E. coli. We credit the use of frequently used E. coli codons for the observed high-level expression. The sperm whale myoglobin produced is stable, easily purified to homogeneity, and indistinguishable from commercially available sperm whale myoglobin by optical and magnetic spectroscopic methods.
抹香鲸肌红蛋白是通过插入表达载体pUC19中的一个完全合成的基因在大肠杆菌中表达的。该基因构建为由23个重叠寡核苷酸组成，编码DNA的两条链。与传统的真核基因克隆技术相比，基因合成具有几个优点，它允许引入一个有效的核糖体结合位点、合适的起始和终止序列、用于方便亚克隆和未来诱变的限制性酶切位点，以及用于高表达大肠杆菌基因的常用密码子。从合成基因表达的抹香鲸肌红蛋白作为全蛋白约占总可溶性蛋白的10%，这表明铁原卟啉IX生物合成和辅基掺入在大肠杆菌中这种血红素蛋白的高水平表达中不是限制因素。我们认为观察到的高水平表达归功于使用了常用的大肠杆菌密码子。所产生的抹香鲸肌红蛋白是稳定的，易于纯化至同质，并且通过光学和磁光谱方法与市售的抹香鲸肌红蛋白没有区别。
