Synthetic Gene DataBase
 

Synthetic Gene 1


 
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Field NameNatural GeneSynthetic Gene
SGDB Gene ID21
GenBank Accession
GenBank GI0
Gene NameSsh10Ssh10_syn
Gene Length (bp)0
SpeciesSulfolobus shibataeEscherichia coli
StrainsDSM 5389BL21(DE3) pLysS, BL21(DE3), BL21Star(DE3), BL21Codonplus(DE3)-RIL
CDS
5' End
3' End
Notes
Expression VectorpET15bpET15
Assay MethodsSDS-PAGE and Biocchrome analyzerSDS-PAGE/Northern Blot/mass spectrometry/Biochrome analyzer
ResultsLow expression in BL21(DE3)Lys strain, but enhance expression in BL21(DE3), BL21* and BL21C+ strainsSlight increase in BL21pLysS and BL21C+ strains, but decrease of expression in BL21(DE3) and BL21* strains
Protein FunctionDNA binding protein
Recoding PurposeTo improve expression
Synthesized ByAuthors
Recoding MethodOptimization according to the codon preferences defined in CUTG database
Publication Author(s)Wu, X.; Jornvall, H.; Berndt, K. D.; Oppermann, U.
Corresponding Author
Corresponding AddressDepartment of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm SE-171 77, Sweden.
Publication Year2004
Publication TitleCodon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance
AbstractExpression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.
JournalBiochem Biophys Res Commun. 313(1): 89-96.
SummaryIn this study, codon optimization failed to improve expression of two heterologous genes in E. coli, which led the authors to postulate that it may be due to the reduced mRNA stability after codon optimization. Two genes used in this study are Ssh10 and mtGrx from the archaeon Sulfolobus shibatae and Methanobacterium thermoautotrophicum, respectively. Wild-type and synthetic genes were cloned into pET15b and expressed in four different strains of E. coli, i.e., BL21 (DE3) pLysS, BL21 (DE3), BL21* (DE3), and BL21Codonplus (DE3)-RIL (BL21c+). The yields of Ssh10_wt (wild type) were similar in all strains except for pLysS, which showed relatively low expression. The authors claimed that this was due to RNA instability in the LysS strain. mtGrx_wt levels were similar in all four strains. Synthetic genes (Ssh10_syn and mtGrx_syn ) were designed by removing all rare codons defined by E. coli genome codon usage table retrieved from CUTG database. The secondary mRNA structures of wild-type and synthetic genes were predicted with mfold and the minimal free energy of the most possible structure was used in this study to indicate the stability of mRNA products. Expression of Ssh10_syn in the E. coli showed some decrease in expression in BL21 (DE3) and BL21* and increase in pLysS and BL21c+. mtGrx_syn expression was too low to be detected. The authors speculated that tRNA abundance did not have as much impact on the level of protein expression as RNA secondary structure stability. In the presence of extra rare tRNAs, protein expression did not improve dramatically nor did it improve with the removal of rare codons from the wild-type sequence. Instead, the optimization of the wild-type sequence resulted in the instability of intermediary RNA (especially in mtGrx) and the decrease in protein expression consequently. However, this hypothesis should be tested more vigorously with genes of improved codon usage and mRNA secondary structures.
CommentsmRNA Data: ĶĪG (kcal/mol) wt=-68.1 syn=-62.3
Discussion
PubMed ID14672702
Submitter NameZheng, Yuanpu
Submitter AddressUMBC
Entry ConfirmationNo
 
 

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