E coli bl21 gold competent cells

E. coli JM109 competent cells (Promega) were used for cloning and subcloning procedures. E. coli BL21 Gold competent cells (Agilent Technologies) were used as the source of endogenous cyclic diadenylate phosphodiesterase, or of genomic DNA for PCR amplification of the coding sequence of mature CpdB, and for recombinant protein expression. The commercial strains were stored at -80°C.

The
lifeact-mScarlet sequence was cut out using NdeI and XhoI sites and
inserted into pET24b+ vector (Sigma-Aldrich Co., St. Louis, MO, 39750),
yielding a 6-His Tag on the mScarlet. Construct was electroporated
into E. coli (BL21-Gold Competent Cells) (Agilent
Technologies, Santa Clara, CA, #230130) and plated on LB plates (Sigma-Aldrich
Co., St. Louis, MO, 52062) with kanamycin (Sigma-Aldrich Co., St.
Louis, MO, 70560–51–9). Growing cultures were sequenced
(Sanger Sequencing, Microsynth Seqlab, Göttingen, Ger) and
expressed in 500 mL of LB Medium (Sigma-Aldrich Co., St. Louis, MO,
51208) for 5 h before harvesting. Cultures were spun down, and the
pellets subjected to lysis buffer (50 mM Tris/Cl pH 8.0, 250 mM NaCl,
10 mM β-mercaptoethanol, 1 mM PMSF). lifeact-mScarlet-6His was
bound to Protino NiNTA agarose (Macherey-Nagel, Düren, Ger,
745400.25) and washed (50 mM Tris/Cl pH 8.0, 250 mM NaCl, 10 mM β-Mercaptoethanol,
20 mM Imidazol). NiNTA beads were given into a empty Protino column
(Macherey-Nagel, Düren, Ger, 745400.10) and washed again. A
final elution was done 5 times with 1 mL of elution buffer (50 mM
Tris/Cl pH 8.0, 250 mM NaCl, 10 mM β-mercaptoethanol, 250 mM
imidazol) for 30 min each, and samples were frozen at −80 °C.
Samples were checked with a SDS-PAGE gel.

hSERINC3-specific binders from phage ELISAs were selected based on signal/background ratios^{47 (link)} and sequenced at the University of Chicago Comprehensive Cancer Center DNA Sequencing facility. Unique clones were sub-cloned in pRH2.2 (a gift of S. Sidhu) using the In-Fusion Cloning Kit (Takara) and sequence-verified. Fab expression vectors were then transformed into E. coli BL21-Gold competent cells (Agilent), and Fabs were expressed as described^{47 (link),48 (link)}. Cells were harvested by centrifugation, and the cell paste was frozen until use. Fabs were purified as previously described in refs. ^{47 (link),48 (link)}.