Production and Characterization of TAT-Fused Proteins

Plasmids used were previously described [10 (link), 11 (link)] or constructed as described in (S1 Fig). Briefly, pJM161 consisted of an E. coli-optimized synthetic gene (GeneScript, Piscataway, NJ, USA) encoding TAT-naked mole rat calmodulin (TAT-NMR-CaM) cloned into NdeI and BamHI sites in pET19b (EMD Millipore, USA) with an in-frame stop codon prior to the BamHI site. The encoded TAT-NMR-CaM protein consists of the TAT peptide sequence (YGRKKRRQRRR) N-terminally fused to Heterocephalus glaber (naked mole rat) calmodulin (GenBank: EHB02604.1) [19 (link)]. A vector-encoded 10xHis tag is N-terminal to TAT. Plasmids pJM140 and pJM168, encoding CBS-maltose binding protein (CBS-MBP) and TAT-maltose binding protein (TAT-MBP), respectively, were made by cloning synthetic gene fragments encoding the CBS or TAT peptide sequences into NdeI and BamHI sites in pMAL-c5x (New England Biolabs, Ipswich, MA). The encoded cargo proteins thus have either a CBS or TAT sequence C-terminal to the MBP and a 6x His tag beyond.
E. coli used in this study, BL21(DE3)pLysS was propagated from purchased cells from EMD Millipore (Burlington, MA, USA) or other established supplier.
Baby hamster kidney (BHK) cells were purchased from ATCC (#CCL-10) and cultured in Dulbecco’s Modified Eagles’ Medium with GlutaMAX Supplement (Gibco, USA) and 10% fetal bovine serum.