Bl21 de3 plyss

The Thai-53 strain of M. leprae [21 (link)], maintained at the Leprosy Research Center, National Institute of Infectious Diseases (Tokyo, Japan), was used to prepare M. leprae DNA. Escherichia coli strain TOP-10 (Thermo Fisher Scientific Inc.; Waltham, MA) was used for cloning. E. coli strains Rosetta-gami 2(DE3)pLysS and BL21(DE3)pLysS (Merck KGaA, Darmstadt, Germany) were used for protein expression. The plasmid vector pET-20b(+) (Merck KGaA) was used for the construction of expression plasmids. Relaxed and supercoiled pBR322 DNA (John Innes Enterprises Ltd.; Norwich, United Kingdom) were used for the DNA supercoiling assay and DNA cleavage assay.

Cultivation of strains and procedures for DNA manipulation were performed as previously described for E. coli (Sambrook and Russel, 2001 ) and S. coelicolor (Kieser et al., 2000 ). For plasmid manipulation and protein overproduction we used Escherichia coli strains XL1blue (Bullock et al., 1987 ) and BL21(DE3) pLysS (Merck Millipore), respectively. For mating experiments we used S. lividans strains TK64 and TK54 (Hopwood et al., 1983 (link)) transformed with the required plasmids. Antibiotics were used at the following concentrations: ampicillin 150 μg/ml; kanamycin 50 μg/ml; thiostrepton 25 μg/ml; apramycin 50 μg/ml. Plasmids are listed in Table 1 and primers in Table 2.

Genes for all designs were purchased as DNA fragments from Twist Bioscience, and cloned into pET11 vectors, containing a N-terminal MBP-tag and His-tag as well as a TEV cleavage site, for bacterial expression. Plasmids were transformed into E. coli BL21 (DE3 pLysS) (Merck, #69451–3) and grown overnight in LB media at 37°C. Pre-cultures were diluted 1:50 and inoculated to an OD₆₀₀ of 0.6 in terrific broth (Condalab, #PRO1246.05) at 37°C and expression was induced by the addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG). Cultures were harvested after 18–20 hours at 20°C. Pellets were resuspended in lysis buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 5% Glycerol, 1 mg/ml lysozyme, 1 mM PMSF, and 1 μg/ml DNase) and sonicated on ice for a total of 12 minutes, in intervals of 15 s sonication followed by 45 s pause. The lysates were cleared by centrifugation (48’384 g, 20 min) and purified using a His-Trap FF column on an Äkta pure system (GE Healthcare), followed by size exclusion on a HiLoad 16/600 Superdex 75 column (GE Healthcare) in phosphate-buffered saline (PBS). Protein concentrations were determined by measuring the absorbance at 280 nm on a Nanodrop (Thermo Scientific). The designed proteins were concentrated by centrifugation (Millipore, #UFC900324) to 1 mg/ml, snap frozen in liquid nitrogen, and stored at −80°C.

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.