title: Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli.
abstract: Proteins from hyperthermophilic microorganisms are attractive candidates for novel biocatalysts because of their high resistance to temperature extremes. However, archaeal genes are usually poorly expressed in Escherichia coli because of differences in codon usage. Genes from the thermoacidophilic archaea Sulfolobus solfataricus and Thermoplasma acidophilum contain high proportions of rare codons for arginine, isoleucine, and leucine, which are recognized by the tRNAs encoded by the argU, ileY, and leuW genes, respectively, and which are rarely used in E. coli. To examine the effects of these rare codons on heterologous expression, we expressed the Sso_gnaD and Tac_gnaD genes from S. solfataricus and T. acidophilum, respectively, in E. coli. The Sso_gnaD product was expressed at very low levels when the open reading frame (ORF) was cloned in pRSET and expressed in E. coli BL21(DE3), and was expressed at much higher levels in the E. coli BL21(DE3)-CodonPlus RIL strain, which contains extra copies of the argU, ileY, and leuW tRNA genes. In contrast, Tac_gnaD was expressed at similar levels in both E. coli strains. Comparison of the Sso_gnaD and Tac_gnaD gene sequences revealed that the 5'-end of the Sso_gnaD sequence was rich in AGA(arg) and ATA(Ile) codons. These codons were replaced with the codons commonly used in E. coli by polymerase chain reaction-mediated site-directed mutagenesis. The results of expression studies showed that a non-tandem repeat of rare codons is critical in the observed interference in heterologous expression of this gene. We concluded that the level of heterologous expression of Sso_gnaD in E. coli was limited by the clustering of the rare codons in the ORF, rather than on the rare codon frequency.
authors.affiliation: School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, Republic of Korea.
authors.forename: Seonghun
authors.initials: S
authors.lastname: Kim
authors.forename: Sun Bok
authors.initials: SB
authors.lastname: Lee
mesh_terms: D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D019766:Genes, Archaeal / Q000235:genetics*; D006836:Hydro-Lyases / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D048229:Sulfolobus solfataricus / Q000235:genetics*; D013822:Thermoplasma / Q000235:genetics*; D013997:Time Factors
chemical_list: D003062:Codon; D012333:RNA, Messenger; D006836:Hydro-Lyases; C020576:gluconate dehydratase