Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5′-coding region in Escherichia coli

Cheong, Dae-Eun; Lee, Jihye; Choi, Hyun-Woo; Yoo, SooNam; Lee, Dong-Hyun; Kim, Geun-Joong

doi:10.1007/s10529-019-02733-y

Cited by 2 publications

(7 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, the protein bands corresponding to the selected scvhFGF19 variants were clearly detected. However, almost all expressed proteins formed an insoluble aggregate as an inclusion body (Figure 2a), although mCherry emitted red fluorescence in the screening step; these results were inconsistent with our previous reports [10,11]. We thus speculated that marginal soluble expression of scvhFGF19 was probably associated with structural features such as the two internal disulfide bonds and the long, disordered region at the N-and C-terminus [12].…”

Section: Expression Of Scvhfgf19 Screened From Synonymous Codon Varia...contrasting

confidence: 73%

“…pET24a_hFGF19 was kindly provided by Dr. Lee Jung-Hyun (Korea institute of Ocean Science & Technology, Busan, Korea) and used as a template to amplify the hFGF19 gene by PCR with specifically designed primers having synonymous codons at the 5 -terminal region [9]. pSCT5-mCherry [10] was used for constructing the synonymous codon library. pACYCDuet1 (New England Biolabs, Hitchin, UK) and pQE80L (Qiagen, Hilden, Germany) were used as backbones for the plasmid constructs containing the dsbC and scvhFGF19 genes, respectively, for co-expression in the cytoplasm of E. coli.…”

Section: Bacterial Strains and Chemicalsmentioning

confidence: 99%

“…To express hFGF19 without any change in the amino acid sequence, codons encoding the first ten amino acids except for ATG were randomly substituted with synonymous codons as described previously [10]. From synonymous codon variant (scv) libraries consisting of approximately 2.8 × 10 4 independent clones, 20 scvhFGF19-mCherry-fused variants were preliminarily screened based on red fluorescence intensity, using fused mCherry as a reporter (Figure S1).…”

Section: Expression Of Scvhfgf19 Screened From Synonymous Codon Varia...mentioning

confidence: 99%

“…We have previously reported that the solubility and expression of aggregation-prone and difficult-to-express proteins, respectively, are improved by synonymous codon substitutions in the N-terminal region encoding sequences of the corresponding genes [9,10]. Thus, in this study, we attempted to screen synonymous codon variants of hFGF19 (scvhFGF19).…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in E. coli

et al. 2020

Self Cite

View full text Add to dashboard Cite

Human fibroblast growth factor 19 (hFGF19) is a difficult-to-express protein that is frequently fused with another protein for soluble expression. However, residual amino acids after cleavage with protease represent one of the major problems in therapeutic protein development. Here, we introduced synonymous codon substitutions in the N-terminal region encoding sequence of hFGF19 and co-expressed disulfide bond isomerase (ΔssDsbC) to functionally express hFGF19 without any fusion protein. Synonymous codon substitution significantly increased hFGF19 expression. Subsequent co-expression of ΔssDsbC with a selected variant of hFGF19 (scvhFGF19) further increased the proportion of soluble hFGF19 expression in Escherichia coli XL1-Blue. Both total and soluble scvhFGF19 expression increased remarkably in the alternative host, E. coli Origami 2 with mutated thioredoxin reductase and glutathione reductase. scvhFGF19 purification by anion exchange and heparin affinity chromatography resulted in a yield of 6.5 mg/L under normal induction conditions in flask culture. As such, a high cell density culture is expected to achieve an even higher yield. The biological activities of purified scvhFGF19 were assessed based on its ability to activate ERK1/2 signaling pathway in HepG2 hepatocarcinoma cells. In conclusion, the strategy described here may represent an efficient alternative process for the production of hFGF19 and/or related proteins.

show abstract

Section: Expression Of Scvhfgf19 Screened From Synonymous Codon Varia...contrasting

confidence: 73%

Section: Bacterial Strains and Chemicalsmentioning

confidence: 99%

Section: Expression Of Scvhfgf19 Screened From Synonymous Codon Varia...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in E. coli

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…coli . A protein level enhancement was also observed in the heterologous expression of a flagellin derivative using synonymous mutations . Therefore, modifying a limited number of NCSs using synonymous mutations could become an efficient method for metabolic engineering or protein production.…”

mentioning

confidence: 99%

Rational Design of the N-Terminal Coding Sequence for Regulating Enzyme Expression in Bacillus subtilis

Tong

et al. 2021

ACS Synth. Biol.

View full text Add to dashboard Cite

Synonymous mutation of the N-terminal coding sequence (NCS) has been used to regulate gene expression. We here developed a statistical model to predict the effect of the NCSs on protein expression in Bacillus subtilis WB600. First, a synonymous mutation was performed within the first 10 residues of a superfolder green fluorescent protein to generate a library of 172 NCS synonymous mutants with different expression levels. A prediction model was then developed, which adopted G/C frequency at the third position of each codon and minimum free energy of mRNA as the independent variables, using multiple regression analysis between the 11 sequence parameters of the NCS and their fluorescence intensities. By designing the NCS of the 10 signal peptides de novo according to the model, the extracellular yield of B. subtilis pullulanase fused to each signal peptide was up-regulated by up to 515% or down-regulated by at most 79%. This work provided a candidate tool for fine-tuning gene expression or enzyme production in B. subtilis.

show abstract

Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5′-coding region in Escherichia coli

Cited by 2 publications

References 16 publications

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in E. coli

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in E. coli

Rational Design of the N-Terminal Coding Sequence for Regulating Enzyme Expression in Bacillus subtilis

Contact Info

Product

Resources

About