Surface Plasmon Resonance to Study Biomolecular Interactions Using a Sensor Chip

Protocolo

For the present study, a CVN-small ubiquitin-like modifier (SUMO) fusion protein has been used in enzyme-linked immunosorbent assays instead of CV-N and is suitable for cell-based assays. Recombinant full-length influenza A virus HA H3 protein is obtained commercially (see Table of Materials) or expressed in mammalian HEK293 cell lines and baculovirus-infected insect cells according to standard protocols. Wuhan-1 spike protein is expressed in mammalian HEK293 cells. The synthesis of mon-mannosylated peptide (MM) and di-mannosylated peptide (DM) allows the detection of homogeneous ligands to CVN2 and mono-mannosylated small molecules.

1. Creating CV-N constructs

1. For each of the CVN2 variants and CVN2L0 protein (PDB ID 3S3Y), obtain the gene construct with an N-terminal pelB leader sequence and His-tag in pET27b(+) vector from commercial sources (see Table 1).
2. Obtain CVN2L0 and its variants (V2, V3, V4, and V5; Figure 1A,C) in the background of a CVN2L0 template gene consisting of two distinct DNA sequences for each CV-N repeat.
3. Dissolve the lyophilized plasmid DNA in sterile deionized distilled water (ddH₂O) to a final concentration of 100 ng/µL.

2. Preparation of LB-agar plates with plasmid DNA transformed cells

1. Prepare culture medium LB-Lennox by dissolving 10 g/L of peptone, 5 g/L of yeast extract, and 5 g/L of NaCl in ddH₂O (see Table of Materials), and adjust the pH to 7.4. Perform the transformation into competent E. coli BL21 (DE3) for each variant (V2-V5) by chemical method following a previously published report.
2. Split the solution (900 µL and 100 µL), transfer 100 µL on LB-agar plates (50 µg/mL kanamycin), and gently use a sterile cell spreader. Incubate the agar plates overnight at 37 °C.

3. Cloning

1. Subclone the gene for CV-N into the NdeI and BamHI sites of pET11a (see Table of Materials) for transformation (electroporation) into electrocompetent cells following reference.

4. Site-directed mutagenesis

1. To generate CVN2L0 and mutant CVN-E41A in the background of a CVN2L0 template gene containing two distinguished DNA sequences for each CV-N repeat.
2. Make mutations using a site-directed mutagenesis kit (see Table of Materials) and specific mutagenic primers 5'-gagaaccgtcaacgtttgcgataacagagttcagg-3' and 5'-cctgaactctgttatcgcaaacgttgacggttctc-3' for running the PCR.
   1. Start a series of sample reactions using multiple concentrations of double-stranded DNA (dsDNA) template ranging from 5-50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA template). Keep the primer concentration constant.
      NOTE: The PCR mix and thermal cycling protocol are generally used as described in the instruction manual for the site-directed mutagenesis kit.
3. Add the DpnI restriction enzyme (1 µL, 10 U/µL, see Table of Materials) below the mineral oil overlay. Thoroughly and gently mix reactions, spin down in a table-top microcentrifuge for 1 min, and incubate immediately at 37 °C for 1 h for digesting the parental supercoiled dsDNA.

5. Transformation of bacterial cells

1. Thaw the XL1-Blue super-competent cells (see Table of Materials) gently on ice. To transform each control and sample reaction, aliquot the super-competent cells (50 µL) to a prechilled polypropylene round-bottom tube (14 mL).
   1. Transfer 1 µL of the Dpn I-treated single-stranded DNA (ssDNA) from each control and sample reaction (mutated ssDNA) to separate aliquots of the super-competent cells, which synthesize the complementary strand. Swirl the transformation reactions carefully to mix and incubate the reactions on ice for 30 min.
      NOTE: Before transferring the Dpn I-treated DNA to the transformation reaction, it is recommended to remove any remaining mineral oil carefully from the pipette tip. As an optional control, the transformation efficiency of the XL1-Blue super-competent cells needs to be checked by mixing 0.1 ng/µL of the pUC18 control plasmid (1 µL) with a 50 µL aliquot of the super-competent cells.
2. Apply heat pulse to the transformation reactions at 42 °C for 45 s, and then place the reactions on ice for 2 min.
   NOTE: The applied heat pulse has already been optimized for the mentioned conditions in polypropylene round-bottom tubes (14 mL).
3. Add 0.5 mL of NZY+ broth (containing per liter: 10 g of NZ amine (casein hydrolysate), 5 g of yeast extract, 5 g of NaCl, 12.5 mL of 1 M MgCl₂, 12.5 mL of 1 M MgSO₄, 10 mL of 2 M glucose, pH 7.5, and preheated to 42 °C) and incubate the transformation reactions at 37 °C with shaking at 225-250 rpm for 1 h. Plate the correct volume of each transformation reaction (5 µL from control plasmid transformation; 250 µL from sample transformation) on LB-ampicillin agar plates.
   NOTE: For the transformation controls and mutagenesis, spread cells on LB-ampicillin agar plates having 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, 80 µg/mL) and isopropyl-1-thio-β-D-galactopyranoside (IPTG, 20 mM) (see Table of Materials). Inoculate 50 mL of the cell cultures with a single colony of transformed E. coli cells to purify mutated plasmid DNA for analyses. Mutagenesis is confirmed by DNA sequencing at an external facility.

6. Expression and protein purification

1. For a large-scale culture, inoculate a small amount of LB (containing ampicillin) with a single colony from the transformed plate.
2. Using the overnight culture, inoculate the expression culture with additives, such as 10 mM of MgCl₂, 10 mM of MgSO_4, and 20 mM of glucose, diluting the seed culture to 1/100.
   1. Grow cells with vigorous shaking at 37 °C. Grow cells to an Abs 600 nm between 0.4-0.6 (mid-log phase) before cooling the cells to 20 °C. Induce with 1 mM IPTG and grow overnight.
   2. Then, harvest the cells by centrifuging at 4,000 x g for 15 min at 4 °C, and discard the supernatant with a pipette.
3. Resuspend the cell pellet in phosphate-buffered saline (PBS) buffer and re-centrifuge at 4,000 x g for 15 min at 4 °C. Then, discard the supernatant with a pipette. Resuspend the remaining pellet in 10 mL of lysis buffer and incubate the suspension for 1 h at 37 °C.
   NOTE: Composition of lysis buffer: (50 mM of NaH₂PO₄, 300 mM of NaCl, 2% Triton-X 100, 500 ng/mL of lysozyme, 1 mM of phenylmethylsulfonyl fluoride (PMSF), 1 mM of dithiothreitol, 1 mM of MgCl₂, pH 8, see Table of Materials).
   1. Subject the mixture to two freeze-thaw cycles (-80 °C). Separate soluble and insoluble fractions by centrifugation at 4,000 x g for 15 min at 4 °C and analyze them using polyacrylamide gel electrophoresis (PAGE), in particular, sodium dodecyl sulfate (SDS)-PAGE (Figure 1B, C).
      NOTE: Cells can be lysed in several ways, such as freeze-thaw, sonication, homogenization, enzymatic lysis, or a combination of these methods. Purification from inclusion bodies is recommended for collecting high protein yields.
4. Purify proteins using Ni-NTA column chromatography (see Table of Materials). Load the soluble fraction onto a regenerated Ni-NTA bead column (1 mL/min). Wash the system with TBS buffer (50 mM of Tris, 150 mM of sodium chloride, pH 7.5) before starting a gradient (0-100% 500 mM imidazole in TBS over 60 min) and collecting fractions (1 mL/min). Dialyze the purified proteins for biochemical characterization against 100 mM PBS (Figure 1C, D).
5. Alternatively, use His-Select Ni²⁺ affinity gel (see Table of Materials) in 14 mL tubes to bind and re-suspend his-tagged recombinantly expressed CV-N in buffer solutions with 20 mM imidazole and 250 mM imidazole, respectively. Incubate in batch for at least 30 min.
   NOTE: Apply these semi-purified proteins to a single-use prepacked column for buffer exchange and cleanup of biological samples, for example, carbohydrates and proteins, which can load 1-1.5 mL eluate from immobilized metal affinity chromatography.
6. Transfer the protein solutions to centrifugation tubes with a 10 kDa cut-off filter (see Table of Materials) and concentrate them by centrifuging for 10 min at 4,500 x g and 4 °C. For SPR measurements, exchange the analyte solutions to 10 mM HEPES, 150 mM sodium chloride, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% Tween20, pH 7.4 (HBS-EP(+), see Table of Materials).
   1. Add this SPR running buffer to a dilution factor of 1:10 and centrifuge four times to the initial volume for 10 min at 4,500 x g and 4 °C.
7. Determine the protein concentration at 280 nm using a NanoDrop UV-Vis spectrophotometer (see Table of Materials) based on the calculated extinction coefficient (20,440 M^-1 cm^-1) for the main protein CVN2L0 showing a size of 23,474 Da. Use PBS (100 mM, pH 7.0) or SPR buffer as a blank, and measure the protein concentration at three dilution steps (1:1, 1:10, and 1:100).

7. SPR spectroscopy

1. Use the Dual Channel SPR system (see Table of Materials) with running buffer HBS-EP(+) and 10 mM glycine HCl pH 1.5-1.6 as the regeneration buffer. Turn on the instrument, degasser, auto-sampler, and pump, and wash the entire system with ddH₂0 for 1 h. Place ready-to-use running buffer in a separate bottle.
2. Drop immersion oil onto the detector and mount a glass sensor chip (see Table of Materials) coated with a thin gold film and on the upper side functionalized with carboxymethyl dextran hydrogel directly onto the detector below the three-port flow cell. Fix the setting by pulling down the handling.
   NOTE: C19RBDHC30M 200 nm streptavidin derivatized carboxymethyl dextran hydrogel with a medium density of biotinylated severe acute respiratory syndrome coronavirus-2 RBD protein, is a ready-to-use sensorchip with the pre-immobilized ligand.

8. SPR binding assay for CV-N binding to HA, S protein, and RBD

1. Immobilize the proteinaceous ligands to sensor chips following the steps below.
   1. Open a run table by clicking on Form in the menu bar and Run Table Editor in the integrated SPRAutoLink software (see Table of Materials). Choose and click on BASIC_Immobilization from the list of available run tables and follow the steps of the experimental procedure on the computer screen. The respective Sample Editor used is shown in the right upper corner.
   2. Click on Sample Set Editor in the Form section to fill out the reagents list for two racks placed in the autosampler for further analyses. Click on Autosampler Direct Control as a "Tool" in the menu bar to bring the racks forward or back home. Choose 4 °C as the operating temperature.
      NOTE: The SPR software allows for "SPR Instrument Direct Control" and "Pump Direct Control" via selecting the corresponding tools, as well as autosampler-handling, and by clicking on Form; also Run Table Editor, Data-Plot, or Post-Processing can be chosen to perform data analysis. Files are directly saved in the default directory and exported as scrubber.files from the Post-Processing window.
2. Start the pump to infuse ddH₂0 by clicking on Tools and Pump Direct Control and record data by clicking on SPR Instrument Direct Control, and each time Start in the newly appearing windows. Put the coupling reagents (step 8.3) in 300 µL vials, put them into the autosampler racks, and start the run table by clicking on Run.
   NOTE: Chip surfaces are either conditioned with 10 mM glycine buffer pH 9.0 or may have been flushed with 1 M sodium chloride, 0.1 M sodium borate buffer pH 9.0 to condition carboxyl derivatized chip surface for EDC/NHS activation mix.
3. For this simple protein-protein interaction, use the CMD500D chip (see Table of Materials) to generate a micro-refractive index unit (µRIU) = 2500 - 3000 flow cell with immobilized HA and µRIU = 400 flow cell with spike protein. At an infuse flow rate of 15 µL/min, inject an aqueous and equal mixture of 0.4 M N-ethyl-N'-(dimethylamino propyl) carbodiimide hydrochloride (EDC*HCl) and 0.1 M N-hydroxysuccinimide (NHS) by applying the following sequential steps.
   1. Refill pump refill at 25,000 µL/min, perform baseline adjustment for 30 s, inject 90 µL of sample activation solution (EDC/NHS) over 6 min contact time, and then hold for another 5 min.
   2. Repeat this cycle after baseline running for 1 min at 10 µL/min on only the left flow cell (blue) to inject and immobilize chemically synthesized peptides, HA, and spike protein at 20 µg/mL, and allow for the subsequent baseline adjustment with ddH₂O for 1.5 min before quenching the activated chip surface with 1 M ethanolamine HCl pH 8.5.
4. Switch the tubes from the liquid sampler to the degasser from ddH₂0 into the bottle with HBS-EP(+) (Figure 2).
5. Analyze the SPR sensorgrams.
   1. To perform kinetic studies, use various analyte concentrations (10^-5-10^-8 M), with a regeneration step after each injection and blank measurements after different analytes. Change the flow rate to 10 µL/min and start injections for 4 min contact time, then 5 min baseline generation, and two regeneration steps of 2 min each with an interval of 30 s.
   2. Inject the buffer solution for blank measurements whose sensorgrams are subtracted from sample runs to normalize different protein concentrations.
6. Click on Form, scroll down, and change to "Post-Processing" by clicking this operation mode. Click on Add to select binding curves generated over time in the Data Plot Form for each flow cell, and export the overlay as a scrubber file (.ovr). Click on File to open the file saving options. Obtain response curves by aligning left and right curves and subtracting signals of the second reference channel from those of the ligand channel.
   NOTE: Data is operated in "Post-Processing" by defining sensorgrams computationally. It is put into an overlay of sensorgrams from left and right flow cells, or represented as sensorgrams from the difference of both channels.
7. Clean the entire fluidics with 50-100 mM glycine buffer pH 9.5, water, and 20% ethanol before and after binding measurements to remove traces of salt or any protein contamination, or more stringent, with 0.5% SDS and glycine.
   NOTE: To prevent instrument damage, it is recommended to check the mechanical stability of the glass chip before re-inserting the chip cartridge into the instrument if chips have been stored under a buffer or at 100% humidity.

Table 1: Kinetic data obtained from SPR sensorgrams for the binding of CVN2L0 and Cys-Cys bond variants V2, V4, and V5 to HA using a Langmuir 1:1 binding model. K_D [M] = k_off/k_on or k_d/k_a. All data is generated at 25 °C in HBS-EP(+) buffer.

Analyte	kon [M-1*s-1]	koff [s-1]	KD
CVN2L0	5.1 e3	1.3 e-3	255 nM
V2	4.0 e3	1.1 e-3	275 nM
V4	1.2 e3	1.3 e-3	11 µM
V5	1.2 e3	5.8 e-2	5 µM

Resultados

Figure 1: CV-N sequences and expression. (A) CVN2 without a linker between each CV-N repeat (101 amino acids each) and four disulfide bridges are expressed in the pET11a vector in E. coli. (B) Expressions of two independent colonies for CV-N (monomer) and CVN2 (dimer). (C) Disulfide bond variants are purified and analyzed on SDS...

Materiales

Name	Company	Catalog Number	Comments
Äkta primeplus	Cytiva
Amicon tubes	Merck	C7715
Ampillicin	Sigma-Aldrich	A5354
Beckmann Coulter Cooler Allegra X-30R centrifuge	Beckman Coulter	B06320
Cell spreader	Sigma-Aldrich	HS86655	Silver stainless steel, bar L 33 mm
Custom DNA Oligos	Sigma-Aldrich	OLIGO
Custom Gensynthesis	GenScript	#1390661	Cloning vector: pET27b(+)
Cytiva HBS-EP+ Buffer 10, 4x50mL	Thermo Scientific	50-105-5354
Dionex UlitMate 3000	Thermo Scientific	IQLAAAGABHFAPBMBFB
Dpn I restriction enzyme (10 U/μL)	Fisher Scientific	ER1701
DTT	Merck	DTT-RO
EDC	Merck	39391
EDTA	Merck	E9884
Eppendorf Safe-Lock Tubes	Eppendorf	30120086
Eppendorf Safe-Lock Tubes	Eppendorf	30120094
Eppendorf Minispin and MiniSpin Plus personal microcentrifuge	Sigma-Aldrich	Z606235
Ethanol	Merck	51976
Ethanolamine HCl	Merck	E6133
Falcon 50mL Conical Centrifuge Tubes	Fisher Scientific	14-432-22
Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap, Sterile, 25/Pack	Corning	352057
Glucose	Merck	G8270
Glycine HCl	Merck	55097
HA H3 protein	Abcam	ab69751
HEPES	Merck	H3375
His-select Ni2+	Merck	H0537
Imidazole	Merck	I2399
IPTG	Merck	I6758
Kanamycin A	Sigma-Aldrich	K1377
Kromasil 300-5-C4	Nouryon
LB agar	Merck	52062
LB agar	Merck	19344
LB Lennox	Merck	L3022
Lysozyme	Merck	10837059001
Magnesium chloride	Merck	M8266
Magnesium sulfate	Merck	M7506
NaH2P04	Merck	S0751
NanoDrop UV-Vis2000c spectrophotometer	Thermo Scientific	ND2000CLAPTOP
NaOH	Merck	S5881
NHS	Merck	130672
NZ amine (casein hydrolysate)	Merck	C0626
PBS	Merck	806552
PD MidiTrap G-10	Sigma-Aldrich	GE28-9180-11
Peptone	Merck	70171
pET11a	Merck Millipore (Novagen)	69436
PMSF	Merck	PMSF-RO
QIAprep Spin Miniprep Kit (1000)	Qiagen	27106X4
Reichert Software Package Autolink1-1-9	Reichert
Reichert SPR SR7500DC Dual Channel System	Reichert
Scrubber2-2012-09-04 for data analysis	Reichert
SDS	Merck	11667289001
Site-directed mutagenesis kit incl pUC18 control plasmid	Stratagene	#200518
Sodim chloride	Merck	S9888
Sodium acetate Trihydrate	Merck	236500
SPR sensor chip C19RBDHC30M	XanTec bioanalytics	SCR C19RBDHC30M
SPR sensor chip CMD500D	XanTec bioanalytics	SCR CMD500D
Sterilin Standard 90mm Petri Dishes	Thermo Scientific	101R20
TBS	Merck	T5912	10x, solution
Triton-X100	Merck	T8787
Tryptone	Merck	93657
Tween20	Merck	P1379
Vortex-Genie 2 Mixer	Merck	Z258423
X-gal	Merck	XGAL-RO
XL1-Blue Supercompetent Cells	Stratagene	#200236
Yeast extract	Merck	Y1625