Experiments

Protocol

DNA Cloning Methods

PCR

PCR (Polymerase Chain Reaction) is a technique used to amplify a template DNA by cycling through denaturation, annealing, and extension phases with a thermostable DNA polymerase.

1. Set up assembly reactions in PCR-tubes for a total volume of 50µL as follows:
   Typical composition of a PCR reaction.

   Component	Volume/Amount
   Template DNA	1 µL
   Forward Primer	2 µL
   Reverse Primer	2 µL
   High-Fidelity DNA Polymerase	0.5 µL
   2x phanta buffer	5 µL
   dNTP Solution Mix (10 mM)	1 µL
   Nuclease-free water	to 50 µL

2. Mix gently and spin down.
3. Place PCR-tubes in thermocycler and run the following program:
   Table 8: Thermocyler program for a PCR.

   Step	Temperature	Time	Return to step	Passes total
   Initial Denaturation	98°C	30 s		1
   Denaturation	98°C	10 s		25 - 40
   Annealing	60 - 72 °C	15 s		25 - 40
   Extension	72°C	60 s/kb	2.0	25 - 40
   Final Extension	72°C	5 min		1
   Hold	4°C	∞		1

4. Store at 4 °C until further use.

Agarose Gel Electrophoresis

Agarose gel electrophoresis is used to separate DNA fragments of different nucleotide lengths from each other.

1. Depending on the nucleotide lengths of the DNA fragments, gels with a different agarose concentration should be used:
   Agarose concentrations for different nucleotide lengths.

   Nucleotide Length	Agarose Concentration
   600 - 50000 bp	< 1%
   400 - 8000 bp	1%
   100 - 2000 bp	2%
   25 - 1000 bp	3%

2. Add agarose to TAE buffer in the desired agarose concentration and heat in the microwave until the agarose is completely dissolved (be careful to not let the agarose boil over).
3. Pour gel and wait for 15 - 30 min until the gel becomes firm.
4. Mix the product with 5 µL 10xDNA Loading Buffer and load each mixture per tube
5. Run gel at 180 volts for 30-45 min
6. Visualize gel under UV or blue light (depending on the staining agent used).

Purification of DNA fragments from agarose gels

The procedures should strictly follow the instructions of PureLink Quick Gel
Extraction Kit (ThermoFisher Scientific) provided as bellow.

Gibson assembly

Gibson assembly is a molecular cloning strategy used to assemble multiple DNA fragments with overlapping overhangs. In contrast to classical restriction cloning, gibson assembly does not require restriction sites for assembly.

1. Design DNA fragments with 15 - 20 bp overhangs.
2. Amplify DNA fragments via PCR.
3. Set up Gibson assembly reaction mix:
   Typical composition of a gibson assembly reaction.

   Component	Single Fragment Assembly
   vector	final concentration 3 pmol
   fragment	final concentration 6 pmol
   Gibson Assembly Master Mix (2X)	5 µL
   ddH2O	to 10 µL

4. Incubate the reaction mix at 50 °C for 10 min.

Transformation of BL21 (DE3) competent E.coli cells

1. Thaw cells on ice for 5 min.
2. Add 1 - 2 µL of plasmid DNA to each tube of cells and incubate for 30 min on ice. Do not mix.
3. Heat-shock cells at 42 °C for 60 sec.
4. Place on ice for 3 min.
5. Add 900µL LB liquid medium and recover cells for 45 min at 37 °C while shaking.
6. Centrifuge for 4000 rpm for 4 min.
7. Discard supernatant by decanting and resuspend cell pellet in remaining supernatant.
8. Plate 75 µL supernatant on LB agar plates containing 50 µg/mL kanamycin.
9. Incubate the cells in an incubator at 37℃ for 15 hours.

Standard Binder Expression & Analysis

Protein Expression

1. Select specific monoclonal colony and shake it in 5 ml LB liquid medium containing 50 µg/mL kanamycin overnight
2. Inoculate 500 mL LB liquid medium containing 50 µg/mL kanamycin with basically all the bacteria solution
3. The cell culture was grown at 37 °C while shaking until the optical density at 600 nm reached 0.6.
4. Expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM at 16◦C overnight

Cell Lysis

1. Transfer the bacteria solution to 500ml centrifuge tube and harvest cells by centrifugation (4500rpm, 15min, 16°C).
2. Discard the supernatant and resuspend the sediment with 35mL lysis buffer(50mM Tris-HCl (pH=8), 300mM NaCl).
3. The bacteria solution is lysed by high pressure homogeniser at 800 bar for 2.5min at 4 °C.
4. Clear the lysate by centrifugation at 18000 rpm for 45 min at 4 °C, then retain the supernatant

Purification of His-tagged Binders

1. Buffer Preparation

Prepare the following buffers and store them at 4°C. The concentrations provided are typical starting points and may require optimization for the specific protein of interest.

• Lysis/Binding Buffer: 50mM Tris-HCl (pH=8), 300mM NaCl
• Wash Buffer: 20 mM imidazole solution
• Elution Buffer: 300 mM imidazole solution

2. Column Preparation

1. Gently swirl the bottle of Ni-NTA agarose slurry to ensure it is fully resuspended.
2. Transfer an appropriate volume of the slurry to an empty gravity-flow column. The amount of resin depends on the expected yield of the protein; a 1 mL bed volume can typically bind 5-10 mg of a His-tagged protein, but this is protein-dependent.
3. Allow the storage buffer (typically 20% ethanol) to drain from the column by gravity.
4. Equilibrate the resin by washing it with 5-10 column volumes (CV) of Lysis/Binding Buffer. Let the buffer drain completely through the column. This step adjusts the pH and prepares the resin for protein binding.

3. Protein Binding

1. Carefully apply the clarified lysate to the top of the equilibrated Ni-NTA resin bed.
2. After sufficient resuspending, transfer the mixture to a new 50 ml centrifuge tube and incubate the lysate with the resin by mixing gently on a rocker for 60 minutes at 4°C before being loaded onto the column.
3. Allow the lysate to flow through the column by gravity.

4. Washing

1. Once the entire lysate has passed through the column, wash the resin with 8-15 CV of Wash Buffer.
2. This step removes non-specifically bound proteins. Continue washing until the absorbance of the flow-through at 280 nm (A280) returns to baseline.
3. Collect the wash fractions for analysis by SDS-PAGE to confirm that the target protein is not being prematurely eluted.

5. Elution

1. Apply the Elution Buffer to the column. The high concentration of imidazole will compete with the His-tag for binding to the Ni-NTA resin, thereby displacing and eluting the target protein.
2. Collect the eluate in separate fractions (e.g., 0.5-1.0 CV per fraction). This allows for the collection of the most concentrated and pure protein fractions, which typically elute first.
3. Analyze the collected fractions for protein content using a protein assay (e.g., Bradford) and for purity using SDS-PAGE. Pool the fractions that contain the purified protein of interest.

SDS-PAGE

Gel and Apparatus Preparation

• Remove the precast gel cassette from its packaging.
• Rinse the cassette with deionized (DI) water.
• Carefully remove the comb from the top of the gel cassette.
• Remove the sealing tape from the bottom of the cassette to allow ion flow during electrophoresis.
• Install the cassette into the electrophoresis tank (cassette dam may be required for a single gel setup) according to the manufacturer's instructions.
• Fill the inner (upper) and outer (lower) buffer chambers with 1x MOPS Running Buffer. Ensure the buffer in the inner chamber covers the wells completely.
• Use a pipette to gently rinse the wells with running buffer to remove any residual storage buffer and dislodge air bubbles.

Sample Preparation

• Combine 20 μL of the protein sample with 5 μL 5x SDS-PAGE Sample Loading Buffer.
• Heat the mixture at 100°C for 10 minutes for complete denaturation and reduction.
• Centrifuge the samples for 1-2 minutes at high speed to pellet any insoluble material.

Electrophoresis

• Carefully load the prepared samples and a molecular weight marker into the wells of the precast gel. The maximum loading volume for a 15-well GenScript gel is typically 25 µL.
• Secure the lid on the electrophoresis tank and connect the electrodes to the power supply (connect red to red and black to black).
• Run the electrophoresis. For ExpressPlus™ gels, a constant voltage of 120-140 V is recommended.
• Continue the run until the bromophenol blue dye front reaches the bottom of the gel, which typically takes 40-50 minutes.
• Once the run is complete, turn off the power supply and disconnect the electrodes.

Staining and Destaining

• Disassemble the electrophoresis unit and carefully open the gel cassette using a gel knife or spatula.
• Transfer the gel into a clean container with ddH2O and rinse briefly.
• Submerge the gel in coomassie brilliant blue dye and incubate with gentle agitation for 30-60 minutes.
• Pour off the stain and add Destaining Solution.
• Agitate gently, replacing the destaining solution periodically until the protein bands are sharp against a clear background.

Mammalian Cell Protocols

Cell Maintenance

This is a basic protocol for splitting Human Embryonic Kidney 293T (HEK293T) cells to maintain the culture throughout the week, with excess cells reserved for future experiments and plating. Make sure to work in a sterile environment to avoid contamination of the cells.

1. Warm reagents to 37°C and prepare complete DMEM supplemented with 10% FCS and 1% P/S:
   Table 15: Composition of complete DMEM supplemented with 10% FCS and 1% P/S.

   Reagent	Volume [mL]
   Dulbecco's Modified Eagle Medium (DMEM)	445.0
   Fetal Calf Serum (FCS)	50.0
   Penicillin/Streptomycin (P/S)	5.0

2. Take cells out of the incubator and aspirate the old media from the plate or flask.
3. Wash with Dulbecco's Phosphate Buffered Saline (DPBS).
4. Add trypsin and incubate at 37°C until cells start to detach.
5. Add DMEM to neutralize the trypsin.
6. Count cells or decide on the appropriate dilution based on confluence.
7. Dilute with DMEM until the desired concentration is reached.
8. Seed cells onto a new plate or flask, gently shuffle to ensure even dispersal, and return cells to the incubator.

Transfection

Transfection refers to the uptake of exogenous DNA into eukaryotic cells.

1. Ensure a 300 mL suspension cell culture is ready for transfection, with a target density of 2.4–2.5 x 10⁶ viable cells/mL
2. Prepare Solution A (DNA): add 300 µg of plasmid DNA to a sterile 15 mL conical tube and adjust the total volume to 5.0 mL with serum-free medium. Mix gently by inverting the tube.
3. Prepare Solution B (PEI): add 950 µL of PEI reagent to a separate sterile 15 mL conical tube and adjust the total volume to 5.0 mL with serum-free medium. Mix gently by inverting the tube.
4. Incubate both Solution A and Solution B at room temperature for 3 minutes.
5. Add the 5.0 mL of Solution B (PEI) to Solution A (DNA). Immediately mix by gentle pipetting.
6. Incubate the final mixture at room temperature for 30 minutes to allow for the formation of transfection complexes.
7. Aseptically transfer the entire DNA-PEI complex solution into the cell culture flask while gently swirling the flask.
8. Place the flask in an orbital shaker incubator and incubate cells at 37 °C and 5% CO2 for 2 - 3 days.

Materials & Methods

GZMK expression and purification

The gene encoding GZMK was cloned into the pHL-sec vector with a flag tag on the C terminal and a secretion signal tag fused to an enterokinase cleavage site at the N terminus according to previous researches. For GZMK production, the expression vector was transiently transfected into FreeStyle 293-F cells using PEI. One day after transfection, add sodium butyrate to the cell culture system to a final concentration of 5 mM. Fpur days after transfection, the GZMK proenzyme was purified from filtered cell supernatants by affinity chromatography based on anti-Flag affinity beads. The eluent was subject to enterokinase cleavage at 16℃ for 24h(add 1μL enterokinase for 10μg target protein, also add calcium chloride to a final concentration of 2 mM). Finally, GZMK in its active form is purified through gel filtration chromatography.

Determination of GZMK activity

1. Enzymatic Activity Assay Using Z-Lys-SBZL

The enzymatic activity of GZMK was measured in a 100 μL reaction volume. The reaction mixture consisted of 0.1 μM GZMK, 1.0 mM Z-Lys-SBZL substrate, and 1.1 mM DTNB in a buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.01% (v/v) Triton X-100. The substrate and DTNB were pre-dissolved in DMSO, ensuring the final concentration in the assay remained below 5% (v/v). Following reaction initiation, the change in absorbance at 405 nm was monitored every 20 seconds for 30 minutes using a microplate reader.

2. Enzymatic Activity Assay Using a FRET Peptide Substrate

This assay was performed in a 50 μL reaction volume with buffer and GZMK concentrations identical to the previous method. The reaction was initiated by adding the FRET peptide substrate (DABCYL-GDGRSIMTE-EDANS) to a final concentration of 500 μM, with the final DMSO concentration kept below 5% (v/v). Fluorescence was monitored for 30 minutes (read every 20 seconds) on a microplate reader, with excitation and emission wavelengths set to 340 nm and 490 nm, respectively.

3. GZMK Enzyme Kinetics Analysis

To determine enzyme kinetic parameters, the FRET assay was performed with a range of substrate concentrations (500, 250, 125, 60, 30, 15, 7.5, 3.75, 1.875, and 0.9375 μM). The initial velocity (v0) at each concentration was determined from the slope of a linear regression of the data points from 4 to 20 minutes. Finally, the initial velocities were plotted against substrate concentrations and fitted to the Michaelis-Menten equation using non-linear regression in GraphPad Prism to calculate Km and Vmax.

Surface plasmon resonance (SPR) experiments

SPR experiments were performed on the Biacore 8K instrument. GZMK in its active form was immobilized using the Series S Sensor protein A chip (Cytiva). Increasing concentrations (0.156 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) of various binders were flowed over the surface for single-cycle kinetic experiments. The surface was regenerated in 10mM glycine pH 1.0. The experiments were performed at 4°C, using a running buffer containing 1xPBS, 500mM NaCl, 0.05% v/v Tween-20, and 3mM EDTA. For affinity screen experiment, 32 binder solution was adjusted to the same concentration (6.7 μmol) and then repeated the above procedures sequentially to screen for binders with obvious affinity.

Screening of small molecule inhibitors

Small molecule screening experiments were conducted in 50 μL reaction systems, each comprising three components: Protein Buffer, substrate solution, and small molecule solution.

Protein Buffer (Pro): 47.5 μL was added per 50 μL reaction system. GZMK solution (22 tubes) was pooled, centrifuged, and the supernatant concentration was determined to be 0.14 mg/mL. Each 40 mL of Protein Buffer contained 680 μL GZMK solution, 8 mL 5x HEPES, 40 μL 100 mg/mL BSA aqueous solution, 40 μL 10% Triton X-100 aqueous solution, and 31.24 mL ddH₂O. For screening six 384-well plates, approximately 110 mL of Protein Buffer was required, and 120 mL (3 × 40 mL) was prepared to ensure complete pipetting. The GZMK concentration in the Protein Buffer was 88.66 nM, resulting in a final GZMK concentration of 84.23 nM in the reaction system.

Substrate Solution (Sub): 2 μL was added per 50 μL reaction system. A 2.5 mM substrate solution in DMSO was prepared by dissolving 10 mg of substrate in 2.731 mL DMSO per tube, with five tubes combined to yield 13.655 mL of solution.

Small Molecule Solution: 0.5 μL of 1 mM small molecule solution was added per 50 μL reaction system.

Experiments were performed using the Bravo liquid handling system for pipetting, mixing, and dispensing. First, 47.5 μL Protein Buffer was mixed with 0.5 μL small molecule solution and incubated for 90 seconds. Then, 2 μL substrate solution was added and mixed, followed by immediate fluorescence intensity measurement in a microplate reader, recorded every minute for 15 cycles.

Binders expression and purification

DNA sequences encoding the proteins of interest were purchased from Atantares and incorporated into plasmids directly. The plasmids were then transformed into BL21(DE3) competent E. coli. The transformation reactions were used to inoculate starter cultures in 5 ml of LB broth, supplemented with 50 mg/L kanamycin. After shaking overnight at 37 °C, the starter cultures were diluted into 5 500 ml of LB with 50 mg/L kanamycin. These cultures were incubated at 37 °C, shaking, until the optical density reached 0.6–0.8, at which point protein expression was induced by the addition of IPTG. The cultures were then further incubated overnight at 16 °C. Cells were harvested by centrifugation for 15 min at 4500 rpm, pellets were resuspended in lysis buffer (50mM Tris-HCl，300mM NaCl, pH 8.0), the cells were lysed by high pressure homogenization and the lysate was clarified by further centrifugation for 45 min at 18000g. The supernatant was passed through Ni-NTA resin in a gravity column and then the resin was washed with 20 column volumes of 20 mM imidazole. Either the His-tagged protein was eluted with 1ml 300mM imidazole twice. The first 1ml of eluent was loaded onto a Cytiva Superdex 75 Increase 10/300 GL gel filtration column equilibrated in 1x PBS buffer. Fractions containing the desired binders were collected and concentrations were estimated spectroscopically by absorbance at 280 nm. For proteins with no tryptophan, tyrosine or cysteine residues, concentrations were approximated by Bradford reagent absorbance at 470 nm in comparison to BSA standards of known concentration.

Preparation of colloidal gold test strips

1. Optimization of Protein-Colloidal Gold Conjugation

Prior to conjugation, the optimal conditions for protein adsorption onto colloidal gold nanoparticles were determined. First, the optimal pH was identified by adjusting 1 mL aliquots of colloidal gold solution to pH values ranging from 6.0 to 9.0 with 1 M Na2CO3. An excess of protein (≥50 µg) was added to each aliquot, followed by a 15-minute incubation at room temperature. The stability of the mixture was then challenged by the addition of 100 µL of 200 mM NaCl solution. The optimal pH was determined as the lowest pH value that prevented salt-induced aggregation, indicated by the retention of the solution's original red color.

Subsequently, using the optimized pH, the minimum protein concentration required for stabilization was determined. A titration series was performed by adding increasing amounts of protein (5-45 µg) to 1 mL aliquots of the pH-adjusted colloidal gold. After a 15-minute incubation, 100 µL of 200 mM NaCl was added to each tube. The lowest protein concentration that successfully stabilized the nanoparticles against aggregation was identified. For all subsequent conjugations, a working concentration corresponding to this minimum amount plus a 10% surplus was used to ensure robust and complete nanoparticle coating.

2. Preparation and Purification of Colloidal Gold-Protein Conjugates

For large-scale conjugate preparation, 20 mL of colloidal gold solution was adjusted to the predetermined optimal pH under constant stirring. The optimized mass of protein was added, and the mixture was allowed to react for 30 minutes at room temperature. To block any remaining active sites on the gold surface and to stabilize the conjugates, 10% (w/v) Bovine Serum Albumin (BSA) was added to a final concentration of 1% (v/v), followed by an additional 30-minute incubation. The final conjugate solution was stored at 4°C overnight.

The conjugates were purified via centrifugation at 12,000 rpm for 20 minutes. The supernatant was discarded, and the pellet was washed sequentially by resuspension and centrifugation, first in a 2% (w/v) BSA solution, and subsequently in a solution of 1% (w/v) BSA in 0.02 M Tris-Buffered Saline (TBS), pH 8.2. The final purified pellet was resuspended in a storage buffer and stored at 4°C until use.

3. Fabrication and Evaluation of the Lateral Flow Assay Strip

The lateral flow assay (LFA) strips were constructed on an adhesive backing card. The purified colloidal gold-protein conjugate solution was dispensed onto the conjugate pad (10 µL per strip) and dried. A solution of capture protein (1 µL) was immobilized onto a nitrocellulose membrane to form the test line. Finally, a sample pad and an absorbent pad were laminated onto the card at the proximal and distal ends of the nitrocellulose membrane, respectively, to complete the assembly. The functionality of the fabricated LFA strips was verified by applying 40 µL of Phosphate-Buffered Saline (PBS) to the sample pad. The results were visually interpreted after a 15-minute development period at room temperature.

Sequence

GZMK expression

Plasmid construction for prokaryotic expression

Plasmid construction for eukaryotic expression

Primer sequences related to plasmid construction

primer-sequence-for-gzmk.csv

▶primer-sequence-for-gzmk.csv

primer	sequence
GZMK-1	tgatgacgatgacaaaATTATTGGTGGTAAAGAAGTAAGCCCTCAC
GZMK-2	ttggtaccgttggtgtGGGGCGGCA
GZMK-3	acaccaacggtaccaaGCACCACCATCACC
GZMK-4	tttgtcatcgtcatcaCCGGTTTCAGCTACGCAAC
GZMK-5	ggatgacgatgacaaaATTATTGGTGGTAAAGAAGTAAGCCCTCAC
GZMK-6	ttggtgccgttggtgtGGGGCGGCA
GZMK-7	acaccaacggcaccaaGCACCATCACC
GZMK-8	tttgtcatcgtcatccACCAAGGCCAAGATAAGTGCGA
GZMK-9	cgatgacgatgacaaaATTATTGGTGGTAAAGAAGTAAGCCCTCAC
GZMK-10	ttggtgccgttggtgtGGGGCGGCA
GZMK-11	acaccaacggcaccaaGCACCATCACC
GZMK-12	tttgtcatcgtcatcgTCACCAGTGGAACCTGGA
GZMK-Flag	CTTATCGTCGTCATCCTTGTAATCGTTGGTGTGGGGC
pcDNA-Flag	GATTACAAGGATGACGACGATAAGTGACTCGAGtctagagggc
pHLcx_FOR	CTAGAGCCTCTGCTAACC
Vector.REV	TTTTGTCATCGTCATCACCGGTTTCAGCTACGC
pHL-R1	TTTGTCATCGTCATCACCGGTTTCAGCTACGC
GZMK-F1	CCGGTGATGACGATGACAAAATTATTGGTGGTAAAGAAGTAAGCCC
Fragment.FOR	CGGTGATGACGATGACAAAATTATTGGTGGTAAAG
Fragment.REV	TTGGTACCGTTGGTGTGGGGCGGCAC
GZMK-R1	CTTATCGTCGTCATCCTTGTAATCGTTGGTGTGGGGCGG
pHL-F1	GATTACAAGGATGACGACGATAAGTAATGATCACTCGAGACTAGTATCGC
pHLcx_REV	CAAGGGGCTTCATGATGT
pET-28a-3	ATACCAACCACCACCACCACCACCACTGAGATCC
vector-F	CTGCGCATATGGCTGCCGCGCG
pET-28a-4	ATTTCCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGG
pET-28a-1	GATATACCATGGAAATTATTGGTGGCAAAGAAGTGAG
pET-28a-2	TGGTGGTGGTTGGTATGCGGCGGCAC

GZMK sequence

gzmk-sequence.csv

▶gzmk-sequence.csv

Protein	sequence
DDDDK-mature GZMK-6His	ETGDDDDKIIGGKEVSPHSRPFMASIQYGGHHVCGGVLIDPQWVLTAAHCQYRFTKGQSPTVVLGAHSLSKNEASKQTLEIKKFIPFSRVTSDPQSNDIMLVKLQTAAKLNKHVKMLHIRSKTSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSRKLCNSQSYYNGDPFITKDMVCAGDAKGQKDSCKGDSGGPLICKGVFHAIVSGGHECGVATKPGIYTLLTKKYQTWIKSNLVPPHTNGTKHHHHHH
GZMK-6His	MEIIGGKEVSPHSRPFMASIQYGGHHVCGGVLIDPQWVLTAAHCQYRFTKGQSPTVVLGAHSLSKNEASKQTLEIKKFIPFSRVTSDPQSNDIMLVKLQTAAKLNKHVKMLHIRSKTSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSRKLCNSQSYYNGDPFITKDMVCAGDAKGQKDSCKGDSGGPLICKGVFHAIVSGGHECGVATKPGIYTLLTKKYQTWIKSNLVPPHTNLEHHHHHH
GZMK-Flag before enzyme digestion	METDTLLLWVLLLWVPGSTGDDDDDKIIGGKEVSPHSRPFMASIQYGGHHVCGGVLIDPQWVLTAAHCQYRFTKGQSPTVVLGAHSLSKNEASKQTLEIKKFIPFSRVTSDPQSNDIMLVKLQTAAKLNKHVKMLHIRSKTSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSRKLCNSQSYYNGDPFITKDMVCAGDAKGQKDSCKGDSGGPLICKGVFHAIVSGGHECGVATKPGIYTLLTKKYQTWIKSNLVPPHTNDYKDDDDK
GZMK-Flag after enzyme digestion	IIGGKEVSPHSRPFMASIQYGGHHVCGGVLIDPQWVLTAAHCQYRFTKGQSPTVVLGAHSLSKNEASKQTLEIKKFIPFSRVTSDPQSNDIMLVKLQTAAKLNKHVKMLHIRSKTSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSRKLCNSQSYYNGDPFITKDMVCAGDAKGQKDSCKGDSGGPLICKGVFHAIVSGGHECGVATKPGIYTLLTKKYQTWIKSNLVPPHTNDYKDDDDK
precursor of GZMK	MTKFSSFSLFFLIVGAYMTHVCFNMEIIGGKEVSPHSRPFMASIQYGGHHVCGGVLIDPQWVLTAAHCQYRFTKGQSPTVVLGAHSLSKNEASKQTLEIKKFIPFSRVTSDPQSNDIMLVKLQTAAKLNKHVKMLHIRSKTSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSRKLCNSQSYYNGDPFITKDMVCAGDAKGQKDSCKGDSGGPLICKGVFHAIVSGGHECGVATKPGIYTLLTKKYQTWIKSNLVPPHTN
mature GZMK-6His	IIGGKEVSPHSRPFMASIQYGGHHVCGGVLIDPQWVLTAAHCQYRFTKGQSPTVVLGAHSLSKNEASKQTLEIKKFIPFSRVTSDPQSNDIMLVKLQTAAKLNKHVKMLHIRSKTSLRSGTKCKVTGWGATDPDSLRPSDTLREVTVTVLSRKLCNSQSYYNGDPFITKDMVCAGDAKGQKDSCKGDSGGPLICKGVFHAIVSGGHECGVATKPGIYTLLTKKYQTWIKSNLVPPHTNLEHHHHHH

Binder expression

binder-sequence.csv

▶binder-sequence.csv

Binder	sequence
1-1	EEHKKKAEQLLEDAKKFKEEGDKEDAKYAAESAIAEAKKLPEYYEEVEKKAKEILS*
1-2	EKEKEVKEYEEDTKKYAEGSSEAAKEDDPSKAEEAKKAAEEQGKKDQENISK*
1-3	MEELKKEAKEIAENGTPEQAAIASSAYWAVEKYKKDPKKAKEYYNYLKSQI*
1-4	SSKKEAEELKKKSEEYKKESEEYKEEAKEYEKEGNTKAAESANKLSEEYEKESEKYEKKAEELEK*
1-5	SEDSKEELEFTKKLAKHYDKETQKAILESLAEQYKELGDEETAKKAEEEAKKAYE*
1-6	ERAKKVREEIERTYKEKTKEEAVDKAYELATKAYQEGDEEAAKEAEREAEKYSK*
1-7	NELKEKTKEYSEKAKEYKKKGDEEKSEAYLQKSESYAWLGGEESEKEYEKIKKEIE*
1-8	KEEEVKELKEKSELAKEYSKEVKEEAEKIYKETGNKQALEKGKELAEDAEKSYKEYEKKAKELEE*
1-9	KIIDDEELAETNKKIEELEKKDEKEAQKLAEELGEKIKENEKKRKEEKEK*
1-10	KLEEGLKWLEEKTAEAIEKGDEENARKYSLAGWESSEKGWDKEETLKYAEELIEN*
1-11	SEEIKKEWKEKVLKGIKEGDEAYQKVIDEALEKDESGNDEETTAIEEGIYEAKKEYK*
1-12	QKELAEKAAEYSIKSYELEKEGKEEESLKYIEEEAKLQRKMDQEGIEEYEEILKEKT*
1-13	SKEESYKEADKAIEEAKKKNYEEAQEIINKQTAEHLEKGDEETARYTTIKGQEVI*
1-14	EKEREEAEEVGRRIREASEEEATKIAGESWQRFLDKGWYKAAELVREYYEEWLKSR*
1-15	DETAKKGEEYYEKAKEYYKEGKEDEATEYYSKAHKYYAKTGNKENQDKVIDDYEKN*
1-16	SSERRQRRLDRLKESLKNAPKDLEELEKEHPELVKKHKSYEELKKELEKGVKKIESGELTDEEAKELTSKITEHMTSIYTYIETTLEKKK*
1-17	SSEYYTEKAKEYSKKGEENKDNPYLAYSYYSRASTYADVAGDKELSEEAEKKAKEAE*
1-18	SYREYAEEAIKKAEESKDEKESEKILEEALEKLEKKYEEYKKEEEKKKEEEE*
1-19	LEEKAKEYYKKGEEYYKKGNKEEAARYYAKAGNEYSKAGKKEEAEKILKKADEVDE*
1-20	EKKKKAEEYKKKSEEYKKKSEELEKKSKEYEEKGDEENSSKSLEESAKYYRESYKYELESEKLEE*
1-21	SEGEKYYKKGKEYEKKGDYKKSSEYYKKSSLKYLEEGNEDNAEKSYEESLKSEEKYK*
1-22	DEKKEEAKEYYEDAKEAKKVGNYKTAADYAKAAEELAKEAGDKETAEKAKKLEKEAK*
1-23	STEKGKEYYEKGKEYYKEGNKNEALKYYGKSASYYATSGDDEGYNKSYENAEKTEE*
1-24	SAVEKAKKAAEKLSEKGYEGAGKSGVELAKDAEKAGDTETAEEIAKDTEELAKE*
1-25	SEVEKAKEAAEELNKKGYTGAGNSGYKLAKDAEKAGDTETAKEIAEDTKELAKE*
1-26	LKEKADEYYEKGEKYEKEGNKDEAAEYYLQASNLYSDYGDDENAEKANQKSLDVAK*
1-27	SSSTWSEAVKYSLKLSEKDPEEATELLSKAYEAKEKGDTETIEEQRKKAKEKVKEL*
1-28	SDEEKANEKVVDYGEKKGASTEAIVAGDAEAGKAIEKGKSAEEAAKIGIDKINELT*
1-29	SKRIEEQKKNIEKSKKATEELIKNKEELTEEELEGVKEYSKEVEKAEKELEKEK*
1-30	SAEEYKKKGDEYEKKGDKENAQEYYLEAANKYYEEGKREESRKYAKKAEEL*
1-31	DDVEYLKKEAKETLEISEKYEEKGKKYEKEGDKEKAYLELGLSERYKEAAKRLEESAKEAE*
1-32	EKEKKAKEYKKKSDEYLKKSGEYNDEASKYYKEGKTEEGDKYDEKSKEYEDKSTEYLEKSSELEN*
2-1	MGSSHHHHHHSSGLVPRGSHMSDRIELQKKNLEKSKKATEELIRQKEELTQEELEGVIEYSKVVEKAEKELEKTK
2-2	MGSSHHHHHHSSGLVPRGSHMSKKIKEQKKHLEKSKEALEELIKNKEELTEEDLEGVKRYSKEVEKAAKELEKEK
2-3	MGSSHHHHHHSSGLVPRGSHMSKRIERQKKNIEKSKKATEELIKNKENLTKEELEGVKEYSKIIEKAEKELEKEK
2-4	MGSSHHHHHHSSGLVPRGSHMMKRINEQMKNIEKSKKATEELIENKEELTEEELEGVKEYSKLVEKAEKELLKEV
2-5	MGSSHHHHHHSSGLVPRGSHMEKAREEQKKNIEKSKKATEELIKNKEELTEEELEGVKEYAKTVEKAKKELEKEK
2-6	MGSSHHHHHHSSGLVPRGSHMSKRVEERLKNIEKSKKATEELIKNKEEMTEEELDGVIEYSKIIEKAEKGLLKEK
2-7	MGSSHHHHHHSSGLVPRGSHMSKTIEEQKKNIEESKKATEDLIKNKEELSEAELEGVKEYSKEVENAERELEEEK
2-8	MGSSHHHHHHSSGLVPRGSHMSARIEEQKKNIEVSKKATEELIKNKEILTEEELEGLKEYSKELAKAEKELAKEK
2-9	MGSSHHHHHHSSGLVPRGSHMSKRIENAKKNIEKSKIAEEELIKNKEELTQEEIEGVKEYSKEVEKAEKELEKEK
3-1	MGSSHHHHHHSSGLVPRGSHMEEPEKIYLKRKTLESTDTDPYNSGTSRTSRLRYYYNHETGTVEEYVYGGNKPNKNTYETAEEAAKKYGSTIKTSDLEEYLRNI
3-2	MGSSHHHHHHSSGLVPRGSHMETEREYRTEKEKKKNKNEEIKDGYSNTYRERWAYNNKKGKIEKHVFGGSDQNGNRWNSEEEYKKASI
3-3	MGSSHHHHHHSSGLVPRGSHMWDKSYINEPIVTGYLNTYYRRYAYDKEKGKIVEYTYGGSDTNGNRWDTKEEAEKKTGITI
3-4	MGSSHHHHHHSSGLVPRGSHMERKPDPTEPITQGYSKTYRERWAYNTEKGRWEKVVFGGSDENGNRWETKEEAEKASI
3-5	MGSSHHHHHHSSGLVPRGSHMKKKVERKKTTPRKQGYTKTYRRRWSYNTETGKWEQYIYGGTDENGNRWETKEEAEKKNKEEGKTYYEYTEEI
3-6	MGSSHHHHHHSSGLVPRGSHMMSEELAKLHLKRAKEAKEKGDKENAINYAKAALDSAKEAGDEETEKEAKELIKEVKI

Proteins for Hardware and preparation of colloidal gold test strips

Plasmids Construction

Sequence

protein-for-hardware.csv

▶protein-for-hardware.csv

Protein	sequence
CyPet-SUMO1	MVSKGEELFGGIVPILVELEGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSVMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKANFKARHNITDGSVQLADHYQQNTPIGDGPVILPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSGMSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV
Ypet-Ubc9	MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLLCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEFLTAAGITEGMNELYKGSGMSGIALSRLAQERKAWRKDHPFGFVAVPTKNPDGTMNLMNWECAIPGKKGTPWEGGLFKLRMLFKDDYPSSPPKCKFEPPLFHPNVYPSGTVCLSILEEDKDWRPAITIKQILLGIQELLNEPNIQDPAQAEAYTIYCQNRVEYEKRVRAQAKKFAPS
mRuby2-Ubc9	MGSSHHHHHHMVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMRIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHMALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFAGLGGGMDELYKGSGMSGIALSRLAQERKAWRKDHPFGFVAVPTKNPDGTMNLMNWECAIPGKKGTPWEGGLFKLRMLFKDDYPSSPPKCKFEPPLFHPNVYPSGTVCLSILEEDKDWRPAITIKQILLGIQELLNEPNIQDPAQAEAYTIYCQNRVEYEKRVRAQAKKFAPS*
mEGFP-SUMO1	MGSSHHHHHHMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGSGMSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV*