Ptwist cmv betaglobin wpre neo mammalian expression vector

For the expression of recombinant forms of antibody clones, nucleotide sequences of antibody heavy- and light-chain antibody variable genes were codon-optimized for mammalian expression and synthesized at Twist Biosciences. Resulting gene fragments were directly cloned at Twist Biosciences into the pTwist CMV BetaGlobin WPRE NEO mammalian expression vector (Twist Biosciences) that had already been fused with the heavy chain Fc domain of human IgG1 or Fc LALA variant. Recombinant antibodies were expressed by transient transfection of 293F cells with polyethyleneimine transfection reagent and was grown in expression medium (Freestyle 293 Expression Medium; Thermo Fisher Scientific). The supernatants were harvested after 7 days, filter-sterilized with a 0.4-μm filter, and purified by affinity chromatography using HiTrap MabSelect Sure columns (GE Healthcare Life Sciences). Purified recombinant IgG were used for in vivo studies.

Recombinant EEEV E1 ectodomain (strain FL93–939; amino acids Y1-S409) with the osteonectin leader sequence and a C-terminal 6x his-tag was codon-optimized, synthesized and cloned into the mammalian expression vector pcDNA3.1(+). Recombinant EEEV (strain FL93–939) structural protein genes (capsid-E3-E2–6K-1) were codon-optimized, synthesized and cloned into the mammalian expression vector pcDNA3.1(+) for expression of the WT EEEV structural proteins. Using the WT EEEV structural protein vector, residues D1-L267 of EEEV E2 structural protein were mutated to alanine or alanine residues to serine for expression of the EEEV E2 mutants for alanine-scanning mutagenesis library analyses. Recombinant human anti-EEEV variable genes were synthesized and cloned into a pTwist CMV Betaglobin WPRE Neo mammalian expression vector that was customized to contain isotype-specific constant regions (IgG1, IgA1, or Fab) (Twist Bioscience Inc.). Additionally, for expression of polymeric IgA1, the mouse J chain sequence (Uniprot: P01592) was synthesized a pTwist CMV Betaglobin WPRE Neo mammalian expression vector (Twist).