The variable domains of the humanized Apo-1 antibody (EP2920210B1), the humanized 2H7 antibody (EP2920210B1), the 4G7 antibody (GenBank no.: AJ555479 and AJ555622), and the MOPC-21 antibody (GenBank no.: AAD15290.1 and AAA39002.1) were codon-optimized using the GeneArt GeneOptimizer tool for the transfection of CHO cells (Thermo Fisher Scientific). VH, VL, and scFv sequences were synthesized de novo at GeneArt (Thermo Fisher Scientific). As previously described, the variable domains were inserted into a human IgGγ1sc backbone, which is designed to abolish FcR-binding and complement fixation [24 (link)]. IgGsc molecules were produced in the ExpiCHOTM Expression System (Thermo Fisher Scientific) according to the manufacturer’s instructions and then purified by HiTrapTM MabSelectTM SuRe columns (Cytiva, Freiburg, Germany), before being subjected to preparative and analytical size exclusion chromatography (SEC) using HiLoadTM 16/600 Superdex 200 pg and SuperdexTM 200 Increase 10/300 GL columns (Cytiva), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described [25 (link)]. The generation and purification of Fabsc molecules were described by Nalivaiko and colleagues [23 (link)].
Geneart geneoptimizer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneArt GeneOptimizer is a software tool developed by Thermo Fisher Scientific for the optimization of gene sequences. It is designed to assist researchers in improving the expression and functionality of synthetic genes by analyzing and modifying their codon usage, GC content, and other sequence characteristics.
Automatically generated - may contain errors
Lab products found in correlation
In fusion cloning, by Takara Bio (2 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Expicho expression system, by Thermo Fisher Scientific (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
Ecorv, by Thermo Fisher Scientific (1 mentions)
Pcold gst, by Takara Bio (1 mentions)
Pcold gst vector, by Takara Bio (1 mentions)
Gibson assembly mix, by New England Biolabs (1 mentions)
Gblock, by Integrated DNA Technologies (1 mentions)
Freund s adjuvant, by Merck Group (1 mentions)
6 protocols using geneart geneoptimizer
1
Humanized Antibody Production and Purification
2
Cloning and Expression of Cas13bt Proteins
3
Structural Characterization of DAX-DIX Complex
4
Generating Codon-Optimized Vector for ABE7.10
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To obtain a fully codon-optimized vector for ABE7.10 expression in mammalian cells, the previously described ABE7.10_4.1 plasmid containing a T2A-Venus reporter and a human U6 promoter-driven sgRNA expression cassette20 was digested with PacI and BglII to excise the TadA heterodimer and the 5′ region of SpCas9(D10A). The corresponding codon-optimized sequence (GeneArt Gene Optimizer, Thermo Fisher Scientific, Massachusetts, USA) synthesized as a gBlock Gene Fragment (IDT; Table S2 ) with 20 nucleotide homology arms was inserted using the Gibson Assembly Mix (New England Biolabs, Massachusetts, USA). The new vector was called ABE7.10co_4.1. All of the plasmid-based ABE experiments described here were performed using ABE7.10co_4.1, although for simplicity, we refer to it as ABE7.10. sgRNA cloning was performed as described.20 Briefly, ABE7.10_4.1co was digested with BplI and oligos containing the spacer sequence, and overhangs to the BplI-digested vector backbone were annealed and ligated. All of the constructs were confirmed via Sanger sequencing. Oligos and sgRNAs used for cloning are listed in Table S3 .
5
Antibody Variable Region Cloning
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The variable regions of the D25 and Motavizumab antibodies were cloned into plasmids encoding the human constant heavy and light chain backbone sequences as previously described48 (link). Briefly, variable region sequences were obtained from the PDB database and codon-optimised for CHO cell expression by GeneArt GeneOptimizer (ThermoFisher Scientific), with complementary sequences added to the 5′ and 3′ ends to facilitate In FusionTM cloning (Clontech) into mouse and human constant domain expression scaffolds using mAbXpress heavy and kappa chain vectors, kindly provided by Dr Martina Jones, AIBN, University of Queensland. These constructs were synthesised as a gBlock (Integrated DNA Technologies) and cloned into the mAbXpress vectors linearised by SacI (New England Biolabs).
6
Generation and Reformatting of Anti-Dengue Antibodies
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Generation of the 3E31 mAb has been described previously (Li et al., 2013) . Briefly, BALB/c mice were immunized intraperitoneally with recombinant DIII proteins corresponding a combination of all four serotypes, and Freund's adjuvant (Sigma-Aldrich). After four booster inoculations, the splenocytes from the immunized mice were harvested and fused with NS-1 myeloma cells to generate hybridomas according to published procedures (Che et al., 2005) . After screening by indirect ELISA and immunofluorescent assay (IFA) as described previously (Xu et al., 2006) , the positive hybridoma cells were subcloned by limiting dilution.
To enable antibody reformatting synthetic oligonucleotides encoding the variable regions of antibodies 3E31 and 4E11 were cloned into plasmids containing the human constant heavy and light chain backbone sequences. Variable region sequence of 4E11 was obtained from the PDB database and codon-optimised for CHO cell expression by GeneArt GeneOptimizer (ThermoFisher Scientific), with complementary sequences added to the 5' and 3' ends to facilitate In FusionTM cloning (Clontech) into human constant domain expression scaffolds using mAbXpress heavy and kappa chain vectors, kindly provided by Dr Martina Jones, AIBN, University of Queensland (Jaberolansar et al., 2017; Jones et al., 2010) . Recombinant antibodies were produced in CHO cells as described for DIII above.
To enable antibody reformatting synthetic oligonucleotides encoding the variable regions of antibodies 3E31 and 4E11 were cloned into plasmids containing the human constant heavy and light chain backbone sequences. Variable region sequence of 4E11 was obtained from the PDB database and codon-optimised for CHO cell expression by GeneArt GeneOptimizer (ThermoFisher Scientific), with complementary sequences added to the 5' and 3' ends to facilitate In FusionTM cloning (Clontech) into human constant domain expression scaffolds using mAbXpress heavy and kappa chain vectors, kindly provided by Dr Martina Jones, AIBN, University of Queensland (Jaberolansar et al., 2017; Jones et al., 2010) . Recombinant antibodies were produced in CHO cells as described for DIII above.
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