title: High-level expression of sperm whale myoglobin in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana 61801.
authors.forename: B A
authors.initials: BA
authors.lastname: Springer
authors.forename: S G
authors.initials: SG
authors.lastname: Sligar
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D005818:Genetic Engineering; D006418:Heme; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009211:Myoglobin / Q000235:genetics*; D009841:Oligonucleotides / Q000138:chemical synthesis; D013057:Spectrum Analysis
chemical_list: D003062:Codon; D009211:Myoglobin; D009841:Oligonucleotides; D006418:Heme