Optimizing Recombinant Claudin Expression

cDNAs encoding human genes of CLDN-1 to -25 were amplified from the Mammalian Gene Collection full-length cDNA clone set^{30 (link)} or synthesized using gene synthesis (GeneArt, Regensburg, Germany) (Table S1). Codon optimization of CLDN-5 sequence was performed by using GeneOptimizer (GeneArt) to lower and normalize the extremely high GC content of the synthesized genes^{19 (link)}. DNA fragments of CLDN-5 ECL antigen Antigen1 and Antigen2 were also synthesized. For non-tagged protein synthesis, cDNA fragments were inserted into the pEU-E01-GW vector using Gibson Assembly (New England Biolabs, Beverly, MA). For AGIA tag fusion protein expression, pEU-E01-AGIA-GW vector containing a start codon and an AGIA-tag at the 5′-end of the gateway cassette was used^{31 (link)}. Alanine mutants were prepared using PrimeSTAR Mutagenesis Basal Kit (Takara Bio, Shiga, Japan). Plasmids were purified by NucleoBond Xtra midi kit (Macherey-Nagel, Duren, Germany).