M1R and A35R protein sequences from MPXV strain Zaire79 were reverse-translated into their coding sequences using GenSmart™ Codon Optimization (GenScript). The coding sequences were then synthesized by Azenda Life Sciences and cloned into pUC vectors containing an upstream T7 RNA polymerase promoter and downstream polyA sequences. The A35R extracellular domain with a signal peptide was fused with M1R by a peptide linker. The accuracy of the sequences was confirmed by DNA sequencing.
Gensmart codon optimization
Manufactured by GenScript
Sourced in United States, Japan
GenSmart™ Codon Optimization is a tool developed by GenScript that allows users to optimize the codon usage of a DNA sequence to enhance protein expression in a specific host organism. The core function of this tool is to analyze the DNA sequence and suggest codon substitutions that align with the preferred codon usage of the target host, thereby improving the efficiency of translation and ultimately increasing the yield of the desired protein.
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Lab products found in correlation
5 protocols using gensmart codon optimization
1
Recombinant MPXV Protein Expression
2
Codon Optimization and mRNA Structure Analysis
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For the vaccine mRNA to be efficiently translated by the host cells, codon optimization is important. Therefore, the codons of the final vaccine construct were optimized for efficient expression in human cells using several Codon Optimization Tools; JCat [72 (link)], GeneArt Instant Designer by Thermofisher, GenSmart™ Codon optimization by GenScript (GS), Codon Optimization Tool by Integrated DNA Technologies (IDT). The quality of the optimized codons was analyzed Using Rare Codon Analysis tools by GS. This tool can predict the efficiency of the translation of the mRNA expressed as the codon adaptation index (CAI) value. Also, the presence of any tandem unusual codons can be detected, shown as codon frequency distribution (CFD). Based on these parameters, the best-optimized sequence was chosen for further assessment.
The secondary structure of the mRNA construct was predicted using the RNAfold tool of ViennaRNA Package 2.0 [73 (link)]. Both the minimum free energy (MFE) structure and the centroid secondary structure of the mRNA were obtained from this tool along with their MFE.
The secondary structure of the mRNA construct was predicted using the RNAfold tool of ViennaRNA Package 2.0 [73 (link)]. Both the minimum free energy (MFE) structure and the centroid secondary structure of the mRNA were obtained from this tool along with their MFE.
3
Codon Optimization and Harmonization for Recombinant Protein Expression
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Codon optimization [49 (link)] and codon harmonization approaches [83 (link),84 (link)] were used in parallel to evaluate their effectivity in obtaining properly folded, soluble protein from each of the selected genes tailored for heterologous expression in E. coli. Codon-optimized gene sequences were generated via GenSmart Codon Optimization (GenScript, Piscataway, NJ, USA) online tool following default codon optimization parameters. Codon Harmonizer developed by Claassens et al. [51 (link)] online tool was used to harmonize codon usage frequencies between the prophage host H. thermotrophus NCBI GenBank (RefSeq GCF_004365575.1) and the heterologous expression host E. coli BL21(DE3) NCBI GenBank (GenBank GCA_000022665.2) (accessed in April 2019). Codon Adaptation Index (CAI) and Codon Harmonization Index (CHI) values were calculated for each sequence. Both the codon-optimized and codon-harmonized target protein gene sequences (Supplementary Materials File S1 ) were ordered to be synthesized and delivered pre-cloned in pET-21b(+) (Merck, Darmstadt, Germany) [85 ] vector (GenScript, Leiden, the Netherlands), featuring a C-terminal hexa-histidine tag [86 (link)] to facilitate purification using affinity chromatography.
4
Optimized Antibody Production Protocol
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All VH and Vκ sequences were described previously (24 (link)) and codon optimized using GenSmart™ Codon Optimization (Genscript) online software, followed by synthesis by Eurofin (Japan) to avoid codon usage bias. The genes were transformed into competent E. coli (DH5α) strains (29 (link)) followed by plasmid extraction (Biobasic Pte Ltd) and sub-cloned into pTT5 vector (Youbio) using restriction enzyme sites, as previously performed (22 (link), 24 (link), 25 (link), 30 (link)).
For testing the effects of additional essential amino acid (EAA) in boosting production, Impact EAA (MyproteinTM) were dissolved in water and filtered with 0.2um filter before supplementing the cell culture media following standard transfection protocol. The supernatant were harvested after 14 days and tested for production levels.
For testing the effects of additional essential amino acid (EAA) in boosting production, Impact EAA (MyproteinTM) were dissolved in water and filtered with 0.2um filter before supplementing the cell culture media following standard transfection protocol. The supernatant were harvested after 14 days and tested for production levels.
5
Optimizing αRep4E3 Gene Expression
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We optimized the codon of the αRep4E3 gene using GenSmart Codon Optimization (GenScript) by inserting the αRep4E3 gene into the website and selecting a human-T cell as a host organism. The optimized αRep4E3 DNA sequences were synthesized by Integrated DNA Technologies. Both the non-optimized and optimized codons were used for the SH200 donor plasmid (GeneCopoeia, Rockville, MD, USA) construction.
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