Enrich sec 650

SEC-MALS measurements were carried out with a miniDAWN TREOS detector (Wyatt Technology Corporation) coupled to an Agilent 1260 Infinity HPLC. A total of 240–360 µg protein samples were injected into a size exclusion chromatography column (ENrich SEC 650, Bio-Rad) and continuously run at a flow rate of 0.5 ml min⁻¹ in the buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM DTT, and 0.02% NaN₃. The molecular weight was determined by multi-angle laser light scattering using an in-line miniDAWN TREOS detector and an Optilab T-rEX differential refractive index detector (Wyatt Technology Corporation). Bovine serum albumin (Sigma, A1900) was used for system calibration and the data were analyzed using ASTRA 6 Software (Wyatt Technology Corporation) with the dn/dc value set to 0.185 ml g⁻¹. Experiment was repeated two times with similar observations, and a representative figure is shown.

Tf and Lf were prepared by dissolving holo hTf (Sigma) or holo hLf (Sigma) in 50mM HEPES pH 8.0, 50mM NaCl. Size exclusion chromatography was performed by injecting the proteins into an ENrich SEC 650 column (Bio-Rad) using the NGC Quest 10 chromatography system (Bio-Rad) at 1ml/min. The eluted proteins were collected and concentrated to 50mg/ml using Vivaspin 20 with a 50kDa cut-off (GE healthcare).

The portion of eccD₅ encoding a predicted soluble cytoplasmic domain from residues 1 to 131 (EccD₅1–131) was amplified from M. tuberculosis Erdman genomic DNA by PCR with primers His-eccD5_F1 and His-eccD5_R1 and cloned into plasmid pET28b+ between the NdeI and HindIII restriction enzyme sites to generate pET28-His₆EccD₅, encoding EccD₅1–131 with an N-terminal His₆ tag. The pET28-His₆EccD₅ plasmid was introduced into E. coli BL21(DE3), and the protein was purified by Ni²⁺-nitrilotriacetic acid (NTA) affinity chromatography (Qiagen) as previously described for PPE41-His₆ (37 (link), 38 (link)). Briefly, purified protein was bound to the column under native conditions in 20 mM HEPES buffer, 300 mM NaCl, pH 7.8, and eluted in 20 mM HEPES buffer, 500 mM NaCl, pH 7.8, containing 50 to 150 mM imidazole. Purified His₆EccD₅ was concentrated through a 5-kDa-cutoff Amicon Ultra centrifugal filtration unit (Millipore), and then contaminant proteins were removed by fast-protein liquid chromatography (FPLC) using a BioLogic DuoFlow apparatus (Bio-Rad). Protein was bound to an Enrich Sec 650 (Bio-Rad) column and eluted in phosphate-buffered saline (PBS) using an isocratic flow at a rate of 500 µl/min. Protein which eluted at 0.02 absorbance unit was collected and pooled.

Rosetta2 (DE3) cells (Merck) were transformed with pET vectors encoding His-tagged proteins. The cells were grown at 37 °C in Luria Bertani medium until the optical density at 600 nm reached 0.6 and protein expression was induced by the addition of 0.2–0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C overnight. To purify the His-tagged BmVasa variants, the cells were suspended with His A buffer [20 mM Tris-HCl (pH 8.0), 1 M NaCl, 20 mM imidazole, 1 mM dithiothreitol, and cOmplete ULTRA EDTA-free protease inhibitor (Roche)] and lysed by sonication. The samples were centrifuged and filtered to remove the cell debris, and then loaded onto Ni Sepharose 6 Fast Flow resin (GE Healthcare). The resin was washed with His A buffer and bound proteins were eluted with His B buffer [20 mM Tris-HCl (pH 8.0), 1 M NaCl, 300 mM imidazole, and 1 mM dithiothreitol]. The eluted proteins were dialyzed against dialysis buffer [50 mM Tris-HCl (pH 8.0) and 100 mM NaCl]. After dialysis, proteins were purified by anion-exchange chromatography and gel filtration using HiTrap SP FF (Cytiva) [to purify ΔN, EnrichQ (Bio-Rad) was used for cation-exchange chromatography] and ENrich SEC650 (Bio-Rad), respectively.

Purified NLGN3 ECD (wild-type or R/C mutant) was concentrated to 1.5 or 1.1 g/L, respectively, and applied onto an ENrich SEC 650 (10 × 300 mm) column (Bio-Rad) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The MALS data were collected on a DAWN HELEOS 8+ detector (Wyatt Technology) with an RF-20A UV detector (Shimadzu) and analyzed by the program ASTRA (Wyatt Technology).

For the expression of LtrCas13a, Escherichia coli Rosetta 2 (DE3) was transformed with the pET-LtrCas13a plasmid, and the cells were cultured in a 2.5 L of LB medium containing kanamycin. When the OD₆₀₀ values reached 0.6–1.0, the cells were cooled on ice for 10 min and further cultured at 20 °C for 20 h with 0.1 mM IPTG. Bacterial cells were collected by centrifugation, suspended in 40 mL of buffer B (20 mM Tris–HCl [pH 8.0], 1 M NaCl, 20 mM imidazole, 3 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride), and lysed via sonication (Q500, QSONICA). After centrifugation at 15,000 rpm for 20 min, the supernatant was incubated with Ni–NTA agarose (Qiagen) at 4 °C for 1 h. The mixture was then transferred to an Econo column (Bio-Rad). The resin was washed with buffer C (20 mM Tris–HCl [pH 8.0], 0.3 M NaCl, 20 mM imidazole, and 3 mM β-mercaptoethanol), and the protein was eluted with buffer D (20 mM Tris–HCl [pH 8.0], 0.3 M NaCl, 300 mM imidazole, and 3 mM β-mercaptoethanol). The protein was then loaded onto a HiTrap SP HP column (Cytiva) equilibrated with buffer E (50 mM HEPES–KOH [pH 7.5], 0.3 M NaCl, and 0.5 mM TCEP). The protein was eluted using a linear gradient from 0.3 to 2.0 M NaCl over seven column volumes. It was further purified through size exclusion chromatography (Enrich SEC 650, Bio-Rad) with buffer F (50 mM HEPES–KOH [pH 7.5], 0.5 M NaCl, and 0.5 mM TCEP).