Bl21 star de3

Cloning and plasmid propagation were conducted in chemically competent NEBTurbo cells (New England Biolabs). Expression of recombinant peptides and proteins was performed in chemically competent BL21 Star (DE3) (Thermo Fisher Scientific). Primers and double-stranded DNA fragments were purchased from Integrated DNA Technologies. Media used for bacterial cultures were purchased from Fisher Scientific. Phusion DNA polymerase, and NEB HiFi Master Mix were purchased from New England Biolabs. Sanger sequencing of plasmids was performed by ACGT, Inc. Plasmid DNA was purified using QIAprep spin columns (Qiagen). HisTrap columns were purchased from GE Healthcare. Gels for SDS-PAGE were purchased from Bio-Rad Labs. Protein solutions were concentrated using Amicon Ultra Centrifugal filters (Sigma Millipore). Antibiotics were purchased from GoldBio. All other chemicals were purchased from Fisher Scientific unless otherwise noted. HalA2, HalM2, CylL_S, and CylL_L were expressed and purified as previously described.^{58 (link), 69 (link)}

BL21 Star (DE3) (Thermo Fisher Scientific) and T7 Express (New
England Biolabs) E. coli containing NBB₁₅₆constructs in pNIC28-Bsa4 or pNIC-CH vectors were grown in LB medium (Thermo
Fisher Scientific) supplemented with 50 g/mL kanamycin (Gold Biotechnology)
to an OD₆₀₀ of 0.8–1.2 before induction with 0.42 mM
isopropyl β-D-1-thiogalactopyranoside (IPTG) (Gold Biotechnology) at
18–22°C overnight. NBB ₁₅₆-expressing E.
coli were grown in MOPS-based minimal media supplemented with
¹⁵NH₄Cl and unlabeled glucose or
¹⁵NH₄Cl and ¹³C-glucose (Neidhardt et al., 1974 (link)) for isotope
labeling under the same growth and induction conditions.

For protein expression BL21 Star (DE3) (Thermo Fisher Scientific),
T7 Express, and T7 Express
lysY/I^q (New England
Biolabs) E. coli containing BOK constructs in pRL574 or
pNIC28-Bsa4 vectors were grown in LB medium (Thermo Fisher Scientific)
supplemented with 50 mg/L kanamycin (Gold Biotechnology) to an
OD₆₀₀ of 0.8–1.2 before induction with 1 mM isopropyl
β-D-1-thiogalactopyrano-side (IPTG) (Gold Biotechnology) at
18–22°C overnight. For isotope labeling WT or G35A
NH–PR3CA expressing E. coli were grown in MOPS-based
minimal media supplemented with ¹⁵NH₄Cl and unlabeled
glucose or ¹⁵NH₄Cl and ¹³C-glucose (Neidhardt et al., 1974 (link)) under similar
growth and induction conditions.

E. coli strain BL21 Star (DE3) from Thermo Fisher (Waltham, MA) was grown for 18 hours in 250 mL of Luria-Bertani broth (2.5 g tryptone, 1.25 g yeast extract and 2.5 g sodium chloride) in a shaker flask at 37 °C. Growth medium (5 mL) was taken and centrifuged at 3000 rpm for 2 minutes and decanted. Phosphate-buffered saline (3 mL) was added, the solution was centrifuged and this cleaning process was repeated twice. Then, 350 μL of this solution was mixed with 350 μL of 1% SDS, 0.2 M NaOH, 10 mM EDTA in 50 mM Tris-HCl pH 8 and incubated for 10 minutes with vortexing every 2 minutes. A resuspended Dynabeads MyOne SILANE suspension (50 μL, Thermo Fisher) was added followed by 400 μL of 2-propanol and was then vortexed and incubated for 5 minutes. Dynabeads were collected by a magnet and the supernatant was removed. A solution of 5 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA and 40% 2-propanol (950 μL) was added for rinsing. After vortexing and incubating for 2 minutes the Dynabeads were collected by a magnet and the supernatant removed. A similar rinse step was performed with 950 μL of 50 mM NaCl, 10 mM Tris-HCl (pH 8), 1 mM EDTA and 70% ethanol. Finally, 100 μL of 10 mM Tris-HCl pH 8 was added, mixed and incubated for 2 minutes, the Dynabeads were collected by a magnet, and the supernatant collected.