HIGH-LEVEL EXPRESSION OF SPERM WHALE MYOGLOBIN IN ESCHERICHIA-COLI
HIGH-LEVEL EXPRESSION OF SPERM WHALE MYOGLOBIN IN ESCHERICHIA-COLI
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DOI：
10.1073/pnas.84.24.8961
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发表时间：
1987-12-01
影响因子：
11.1
通讯作者：
SLIGAR, SG
中科院分区：
文献类型：
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作者：
SPRINGER, BA;SLIGAR, SG
将完全合成的基因插入到表达载体pUC19中，在大肠杆菌中表达抹香鲸肌红蛋白。该基因被构建为23个重叠的寡核苷酸，编码DNA的两条链。与传统的真核基因克隆技术相比，基因合成具有许多优势，包括有效的核糖体结合位点、合适的起始和终止序列、方便亚克隆和未来突变的限制性内切酶位点，以及高表达大肠杆菌基因常用的密码子。该合成基因所表达的抹香鲸肌红蛋白为。apprxeq。10%的可溶性蛋白为全蛋白，表明铁原卟啉IX的生物合成和假体群的掺入并没有限制该血红素蛋白在大肠杆菌中的高水平表达。我们将观察到的高水平表达归功于经常使用的大肠杆菌密码子。所制备的抹香鲸肌红蛋白稳定，易于纯化至均匀性，通过光学和磁光谱方法与市售的抹香鲸肌红蛋白难以区分。
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 .apprxeq. 10% of the total soluble protein as holoprotein, 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.