Cloning and bacterial strains
DNA constructs for the expression of Hs.KRAS4b(1-169), Hs.KRAS4b(2-169), Hs.NRAS(1-169), Hs.RAF1(52–192), and Hs.NRAS(1-169)-ITD in the format of His6-MBP-tev-POI (MBP, maltose-binding protein; tev, tobacco etch virus protease recognition sequence; POI, protein of interest) were created by subcloning Entry Clones into pDest-566 (Addgene #11517) using Gateway LR clonase per the manufacturer’s instructions (Thermo Fisher Scientific). Plasmid R714-X01 (renamed from NCBI Reference Sequence: NM_144547.2; Uniprot Q8K592; ref. 12), encoding FLAG-Mm.AMHR2(18–142)-His6, was received from the legacy Tuohy laboratory at the Cleveland Clinic. Nanobody expression plasmids were provided by Matt Hall (NCATS, Bethesda, MD). See Table 1 for details of the proteins encoded by the plasmids used in this work.
V. natriegens was obtained from Synthetic Genomics, Inc. E. coli expression work was performed with strain BL21 DE3 Star™ (Thermo Fisher Scientific (Waltham, MA)), modified to contain a pRare plasmid (CmR) expressing tRNAs (argU, argW, ileX, glyT, leuW, proL, metT, thrT, tyrU and thrU). Our strain is an isolate (TT1) from this parental line that is resistant to a bacteriophage discovered in our lab (unpublished results).
Chemicals and media
Unless noted otherwise, all chemicals were obtained from MilliporeSigma (Burlington, MA). Instant Ocean™ was from Spectrum Brands (Blacksburg, VA). DMSO was from NEB (Ipswich, MA). Brain Heart Infusion (BHI) Dry media was from Thermo Fisher Scientific. BHI broth medium was prepared as per [4] (BHI + v2 salts) but without the addition of MgCl2 (referred to here as BHIv2-Mg). LB-15 agar petri plates were Lysogeny Broth (LB-Miller modified to 15 g/L NaCl) with 2.0% (w/v) agar amended as needed with either 5 µg/mL ampicillin (for initial transformation plates) or 50 µg/mL ampicillin (for colony isolation) for plasmid maintenance. All liquid cultures were amended with 50 µg/mL ampicillin. ZYM-20052 medium [17] was modified to 1.5% (w/v) Instant Ocean™ with no lactose added (referred to here as ZYM-20050-IO), was used for overnight seed growths. TBV2 medium is (per liter) 12 g tryptone, 24 g yeast extract, 15 g NaCl, 0.5% (v/v) glycerol, 2.31 g KH2PO4, and 16.43 g K2HPO4⋅3H2O. The potassium salts are prepared separately in ~ 100 mL dH2O (per liter of final volume), filter sterilized, and added to the other components which had been autoclaved separately.
Preparation of chemically competent cells
Chemically competent V. natriegens cells were prepared as described [4] by Weinstock et al., with modifications. Specifically, a glycerol stock was used to obtain an isolated colony by a T-streak on an LB-15 agar plate (no antibiotics) and incubated overnight at 30°C, MgCl2 was omitted from the BHI broth, and the final pool of competent cells in transformation storage buffer (prepared as described [4]) was amended with filter sterilized glycerol to 10% (v/v).
Transformation of chemically competent cells
A vial of V. natriegens competent cells was retrieved from storage at − 80°C and allowed to thaw on ice. One hundred ng of plasmid DNA (isolated from E. coli DH10B T1 Phage-Resistant cells, Thermo Fisher Scientific) was added to the tube of competent cells, mixed by flicking the tube gently (~ 10 times), and incubated on ice for 30 min. During incubation, 1 mL BHIv2-Mg was added to a culture tube (14 mL round bottom Falcon™ tube, 352059) and warmed to 30°C. The cells/DNA mixture was heat shocked in a 42°C water bath (without shaking) for 30 s and returned to ice for 1.5 min. The one milliliter of warmed BHIv2-Mg medium was added to the cell/DNA mixture, and the transformation reaction was transferred from the microcentrifuge tube back to culture tube and incubated at 30°C for 1 h with agitation at 250 rpm (1” throw). Three hundred microliters of the transformation were plated on antibiotic selection plates (LB-15 agar plate with 5 µg/mL ampicillin, pre-warmed to 30°C) and incubated at 30°C overnight. The next morning a colony was picked from the 5 µg/mL ampicillin plate and struck out on an LB-15 agar, 50 µg/mL ampicillin plate to obtain isolated colonies by T-streak and incubated at 30°C. After ~ 6–8 h, colonies were visible.
Small-scale growth
Fifty milliliters of ZYM-20050-IO with 50 µg/mL ampicillin in a baffled, 250 mL flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated at 30°C for 14–16 hr with shaking at 250 rpm (1” throw). The following morning, the OD600 of the overnight culture was measured and used to inoculate 30 mL of TBV2 medium + 50 mg/mL ampicillin in a 250 mL baffled flask (Optimum Growth™, Thomson, Oceanside, CA) to a calculated OD600 of 0.1. The flask was incubated at 30°C, shaking at 250 rpm (1” throw). The culture was grown to an OD600 of 1.5-2.0 (~ 2hrs) and protein expression induced with 1 mM of IPTG. The culture was grown for another 6–7 hours and cells were harvested using a 50 mL conical tube at 3900 x g for 25 minutes at 4°C. Fifty milliliter E. coli cultures were grown using the Dynamite medium protocol as described previously [17].
Large-scale growth for protein production
To produce two liters of expression culture, a BioFlo 110 (3-liter vessel, New Brunswick) was prepared the day before the planned growth with a dissolved oxygen (DO) probe (with fresh electrolyte) in two liters of TBV2 medium and autoclaved using a liquid 30 min cycle. The DO probe was attached to the head unit and allowed to polarize overnight (per manufacturer’s instructions). Fifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated overnight at 30°C and shaken at 250 rpm (1” throw). The following morning the TBV2 medium in the 3-liter production vessel was completed by adding 200 mL of filter sterilized potassium salts, antifoam (Antifoam 204, added at 0.5 mL/L from a 50% (v/v with water) stock), and 50 µg/L ampicillin. The air flow was set to 3 L/min and the agitation was set to 481 rpm. The minimum target DO was set to 20%, controlled by agitation primarily (agitation range was set to 481–600 rpm) and then by controlling supplied O2 (range set to 0-100%). The overnight culture density was measured by OD600 and used to inoculate the TBV2 medium in the BioFlo 110 to a calculated OD600 of 0.1. The culture was grown to an OD600 of 1.5-2 (~ 2 hr), protein expression induced with 1 mM of IPTG, the culture grown for an additional ~ 4 hr, final OD600 measured, and the culture harvested at 9000 x g for 30 mins at 4°C. When expression constructs included the CRD domain of RAF1, ZnCl2 was added at 300 µM approximately 1 hr before induction of protein expression. Cell pellets were either lysed immediately or stored at -80°C. Two-liter E. coli cultures were grown using the Dynamite medium protocol as described previously [17].
15 N isotopic labeling of RAF1 RBD-CRD (52–192)
Fifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated overnight at 30°C and shaken at 250 rpm (1” throw). The following morning, one liter of ModM9 [18] medium (amended to 4 g/L glucose, 15 g/L NaCl, and 50 mM 15NH4Cl, cat # NLM-467, Cambridge Isotope Laboratories, Inc., Tewksbury, MA) was prepared in a four-liter baffled shake flask. The OD600 of the overnight culture was measured and the amount of overnight culture required to achieve a starting OD600 of 0.1 was aseptically withdrawn, centrifuged (3900 x g for 15 min at room temperature), resuspended with a small amount of the prepared ModM9, added to the remaining ModM9 culture in the four-liter flask, and incubated at 30°C, 250 rpm (1” throw), for a target OD600 of 0.5. At ~ 1 hr prior to reaching that density, the culture was amended to 300 µM ZnCl2. Upon reaching OD600 = 0.5, the protein expression was induced by the addition of 1 mM IPTG and shifted to 25°C for overnight incubation. Cells were harvested by centrifugation (9000 x g, 30 min at 4°C). The protocols for isotopic labeling in E. coli were described previously [19].
Deuterium labeling of KRAS
Fifty milliliters of ZYM-20050-IO in a 250 mL baffled flask was inoculated with an ice chip from a V. natriegens glycerol stock. This seed culture was incubated overnight at 30°C and shaken at 250 rpm (1” throw). The following morning, the OD600 of the overnight culture was measured and used to inoculate a 250 mL baffled flask (50 mL of ZYM-20050-IO working volume) with a starting OD600 of 0.3 and incubated at 30°C, 250 rpm (1” throw), until reaching an OD600 of 1.5 (~ 1 hr). Fifty mL of ModM9-1.5IO D2O (ModM9 adjusted to 1.5% w/v Instant Ocean™ and made with 100% D2O) was added to the flask (for 50% D2O in 100 mL of culture) and the 100 mL resulting culture transferred to a 500 mL baffled flask and the culture grown until OD600 = 1.5 (~ 1 hr). One hundred mL of ModM9-1.5IO D2O was added to the culture (for 75% D2O in 200 mL of culture), transferred to a one-liter baffled flask and the culture grown until OD600 = 1.5 (~ 1.5 hr). The cells were harvested by centrifugation (3900 x g, 15 min at room temperature) and resuspended in 100% ModM9 + 15 g/L NaCl in 100% D2O, and 250 mL aliquots were placed in four, one-liter baffled shake flasks. The cultures were grown to OD600 = 0.5, protein expression induced with the addition of 1 mM IPTG, and the incubator temperature shifted to 25°C for overnight incubation. Cells were harvested by centrifugation (9000 x g, 30 min at 4°C). The protocols for deuterium labeling in E. coli were described previously [20].
Cell lysis
Cells were lysed using a Microfluidics 110-EH (Microfluidics, Westwood, MA) by resuspending the cells in 10 mL of lysis buffer per 1000 OD600 of the final culture and processing at 13,000 PSI (10,000 PSI for E. coli) for two passes. For RAF1 proteins, Benzonase™ Nuclease HC (cat # 71205-3, Millipore Sigma) was added at 250 units/L of culture and RNase (Qiagen) was added at 350 units/L of culture. Clarification of V. natriegens lysates is best achieved using ultra-centrifugation (104,000 x g) for 30 min. Alternatively, high-speed centrifugation (30,000 x g, as used for E. coli) for 90 minutes can be used, but this approach can lead to longer downstream processing times during sample filtration and/or concentrating protein pools for application to preparative SEC columns.
Protein purification
G-Hs.KRAS4b(1-169), GG-Hs.KRAS4b(2-169) and G-Hs.NRAS(1-169) proteins were purified as described [21] (Protein production section, page S-4, in Supporting Information). Hs.RAF1(52–192) was purified as described previously [22]. Nanobody purification was described previously [23]. FLAG-Mm.AMHR2(18–142)-His6 was purified as described (manuscript in preparation).
Intrinsic GTPase measurement.
The intrinsic rates for KRAS and NRAS were determined by the Phosphate Sensor assay [24]. A 2-fold, 8-point standard curve was generated using KH2PO4 with a starting concentration of 6 µM, with the last point being assay buffer (50 mM HEPES, pH 7.3; 150 mM KCl, 1.5 mM MgCl2, 5 mM DTT). RAS proteins (6 µM final concentration) were aliquoted into a 384-microplate and the reaction was initiated by the addition of the Phosphate Sensor (4.5 µM final concentration). The phosphate standards were included in the plate. The plate was sealed to protect from the light and read from the bottom in the BioTek Synergy Neo2 (Ex 430/5, Em 450/5) and read every 2 minutes and 20 seconds for 8 hours at 37°C.
Intact mass
Mass spectrometry was done as previously described [25]. Briefly, samples were diluted to 0.1 mg/ml and 50 µL analyzed via liquid chromatography coupled on-line with mass spectrometry. High-resolution intact protein mass (MS1) spectra were acquired over a 600–2500 m/z window at 120,000 FT resolution (at 400 m/z) with an AGC target value of 3e + 06 and averaging 4 microscans. Spectra were analyzed by MagTran (Amgen Inc.).