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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

Abstract

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Introduction

Monoclonal antibodies are now broadly used for cancer treatment1. Due to the flexibility of antibodies, various antibody-based formats have been actively explored. Compared with monoclonal antibodies, bsAbs have two different antigen binding modules, enabling them to recognize two different targets simultaneously and efficiently trigger the recruitment of immune effector cells to target and kill tumor cells2.

Current recombinant bsAb formats can be generally assigned to two classes: Fc-containing bsAbs and bsAbs without an Fc region. Compared with Fc-containing formats that are mostly produced in mammalian cells, bsAbs without an Fc region have the advantages of smaller sizes, are more readily produced in microorganism expression systems, and can penetrate tumor tissues more efficiently3.

bsAbs without an Fc region are commonly formed by linking individual binding moieties, such as single-chain variable fragments (scFvs) or Fabs3. Without the stabilizing domains, bsAbs based on scFv fragments often have compromised thermal stability, low solubility, or an increased potential for aggregation4,5. In contrast, Fab-based bsAbs are more stable due to the heterodimerization of the CH1 and CL in the native Fab moiety4,6.

Variable domain from heavy-chain-only antibodies (VHHs, also referred to as single domain antibodies) are the active antigen-binding fragment of natural heavy chain antibodies7. VHHs have the characteristics of high affinity, specificity of conventional IgGs8, low immunogenicity, and high yields in bacterial expression9. Compared with Fv fragments, VHHs have higher thermal stability10. Compared with Fab moieties, VHHs have smaller sizes due to the lack of CH1 and CL. Thus, S-Fab, the bsAb format obtained by linking the Fab with a single domain antibody, VHH, was designed and studied for its anti-tumor effects11,12.

In this study, the construction of GPC3-S-Fab by linking the Fab of hGC3313 with an anti-CD16a VHH14 was described. The GPC3-S-Fab can be efficiently produced by periplasmic expression in Escherichia coli (E. coli). Functional studies of GPC3-S-Fab suggested that GPC3-S-Fab is a promising strategy for liver cancer therapy. Thus, the advantages of GPC3-S-Fab over alternative techniques with applicable references to previous studies include easy production and purification, and more stable bsAbs.

Mammalian expression systems and prokaryotic expression systems have been used to express various formats of BsAbs. In contrast to mammalian expression systems, E. coli-based protein expression systems have many benefits, including high yields, low cost and labor-saving, the ease of genetic manipulations, and high transformation efficiency15. For bsAbs expression in E. coli, there are two basic strategies: expression in the cytoplasm and expression in the periplasm between the cytoplasm and outer cell membranes15. Compared to the reducing environment of cytoplasm, the periplasm is a more oxidizing environment, which promotes the correct folding and co-expression of proteins16. Correct folding plays a key role in solubility, stability and function generation of bsAbs. Therefore, a signal sequence pelB was added to the N-terminus of the S-Fab to direct secretion to the periplasm of E. coli17. To ensure correct folding, solubility, thermal stability, and conformational stability, reducing the complexity and the size of an antibody is frequently employed16. The S-Fab format consists of one Fab and one VHH, which is expressed very well in bacterial systems likely due to the simple structure and small size.

GPC3 was chosen in this GPC3-S-Fab bispecific antibody format. Glypican-3 (GPC3) is a member of the heparin sulfate (HS) proteoglycan family that is anchored to the cell surface through glycosylphosphatidylinositol (GPI)18. GPC3 is overexpressed in 70% of hepatocellular carcinoma (HCC) cases, which account for the majority of liver cancers19,20,21,22. Because GPC3 is rarely expressed in normal tissues, GPC3 has been proposed as a potential target for HCC. Multiple mouse mAbs have been produced against GPC3. However, only GC33 exhibited limited anti-tumor activity 22, and it failed to exhibit clinical efficacy in patients. In this study, GPC3-S-Fab was shown to be able to recruit NK cells to kill GPC3 tumor cells14.

To recruit NK cells, anti-CD16 VHH was used. CD16a is a low affinity IgG receptor, expressed mainly on natural killer (NK) cells, macrophages, monocytes and some subtypes of T cells. It is involved in antibody-dependent cell cytotoxicity (ADCC) by NK cells23. Human NK cells can be categorized into two types, CD56-CD16+ and CD56+CD16-. In contrast to CD56+CD16− NK cells, CD56-CD16+ NK cells can release higher levels of perforin and granzyme B and thus present a strong cytotoxicity24. Kupffer cells (KCs), expressing CD16a, are the resident macrophages in liver. Kupffer cells play an important role in the suppression of liver cancer25. Thus, bsAbs targeting CD16a may be a more promising strategy than engaging T cells against liver cancer.

Protocol

All of the procedures including human blood collection were approved by the Sun Yat-Sen University Ethics Committee.

1. GPC3-S-Fab Design Strategy

  1. Design GPC3-S-Fab by linking a Fab of anti-GPC3 (humanized GC3313) with an anti-CD16 VHH14 (Figure 1).
  2. Synthesize and clone the VH-CH1-CD16-VHH and VL-CL into the pET26b and pET21a vectors as previously reported12.

2. Transformation and Culture

  1. Add 100 ng of pET26b containing VH-CH1-CD16 (Figure 1A) and 100 ng of pET21a containing VL-CL (Figure 1A) into 100 µL of competent BL21 (DE3) cells. Mix well and incubate on ice for 30 min. Incubate in a 42 °C water bath for 45 - 90 s, transfer, and incubate on ice for 3 - 5 min.
  2. Add 500 µL of lysogeny broth (LB) medium into a tube containing BL21 (DE3) cells and then incubate at 37 °C, 150 rpm for 45 - 60 min. Spread 100 µL of BL21 (DE3) cells on LB-agar plates containing 50 µg/mL of kanamycin and 100 µg/mL of ampicillin, and then incubate at 37 °C for 12 - 16 h.
    1. Prepare lysogeny broth (LB) medium: 1% wt/vol tryptone, 0.5% wt/vol yeast extract, 1% wt/vol NaCl.
  3. Inoculate cells from a single colony into 5 mL of super broth (SB) medium containing 50 µg/mL of kanamycin and 100 µg/mL of ampicillin, and culture at 37 °C, 220 rpm, overnight. Then, inoculate 1 mL of culture into 100 mL of SB medium containing 50 µg/mL of kanamycin and 100 µg/mL of ampicillin and culture at 37 °C, 220 rpm for 4 h. Then, transfer 10 mL of the culture into 1 L of SB medium containing 50 µg/mL of kanamycin and 100 µg/mL of ampicillin and culture at 37 °C, 220 rpm.
    1. Prepare super broth medium: 3.2% wt/vol tryptone, 2% wt/vol yeast extract, 0.5% wt/vol NaCl.
  4. When the OD600 reaches 0.6 - 0.8, add isopropyl-b-D-thio-galactopyranoside (IPTG) to a final concentration of 0.2 mM. Culture at 16 °C, 180 rpm for 36 - 48 h for periplasmic expression.

3. GPC3-S-Fab Periplasmic Purification

  1. Periplasmic fraction preparation
    1. Collect cells by centrifuging at 4,000 x g, 4 °C for 30 min, discard the medium, and weigh the cells.
    2. Resuspend the cells thoroughly in 4 mL of ice-cold sucrose solution (20 mM of Tris-HCl, pH 7.5; 25% (wt/vol) sucrose and 1 mM of EDTA) per gram of cells, and incubate on ice for 15 min.
    3. Centrifuge at 8,500 x g (table top centrifuge, fixed angle rotor), 4 °C for 20 min. Remove the supernatant (the sucrose fraction) and save it on ice.
    4. Resuspend the pellets in a mix of ice-cold 5 mM of MgCl2 and 1 mM of PMSF (4 mL per gram cells). Add 40 µL of the lysozyme stock (15 mg/mL) per gram, and incubate on ice for 30 min.
    5. Centrifuge at 8,500 x g (table top centrifuge, fixed angle rotor), 4 °C for 20 min. Transfer the supernatant (the periplasmic fraction) and save it on ice.
    6. Combine the sucrose fraction and periplasmic fraction, and centrifuge at 30,000 x g, 4 °C for 30 min. Remove the supernatant and save it in a 50 mL conical tube on ice.
  2. Ni-NTA affinity purification
    1. Resuspend 1 mL of Ni-NTA agarose by mixing thoroughly to achieve a homogeneous suspension, remove 1 mL of Ni-NTA agarose to a fresh 15 mL conical tube, and add 10 column volumes (CV) equilibration buffer (25 mM of Tris-HCl, pH 7.5; 1 M of NaCl) to equilibrate.
    2. Centrifuge at 400 x g for 5 min, and remove the supernatant carefully. Then, add the Ni-NTA agarose into the 50 mL tube containing the sucrose fraction and periplasmic fraction. Rock at 4 °C for 2 h.
    3. Centrifuge the mixture at 400 x g, 4 °C for 5 min. Carefully remove the supernatant to another fresh ice-cold tube as the unbound fraction. Transfer the Ni-NTA agarose into a gravity column.
    4. Add 10 CV of washing buffer (25 mM of Tris-HCl, pH 7.5; 1 M of NaCl; 20 mM, 30 mM or 40 mM of imidazole) to the column, and collect the elute as the washing fraction.
      NOTE: These solutions should be added in order of increasing concentrations of imidazole.
    5. Add 3 CV of elution buffer (25 mM of Tris-HCl, pH 7.5; 300 mM of NaCl; 100 mM, 200 mM, 300 mM or 400 mM of imidazole) to the column and collect the elute as Elution fraction 1, 2, 3, and 4.
      NOTE: These solutions should be added in order of increasing concentrations of imidazole.
    6. Dialysis the eluted fractions in 2 L of PBS (137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, 2 mM of KH2PO4, pH 7.4) using dialysis tubing (12.4 kDa) at 4 °C for 2 h. Check the presence of GPC3-S-Fab by 12% SDS-PAGE and then by Coomassie blue staining.
  3. IgG-CH1 affinity purification
    1. Transfer 1 mL of IgG-CH1 affinity resin into a 50 mL conical tube, and wash the beads with PBS first. Then, add the dialyzed solution containing GPC3-S-Fab after Step 3.2.6. Rock at 4 °C for 2 h.
    2. Centrifuge at 400 x g for 5 min, 4 °C. Carefully remove the supernatant to another fresh ice-cold tube, and transfer the resin to a gravity column.
    3. Add 10 CV of washing buffer (PBS) to the column, and collect the elution as the wash fraction.
    4. Prepare the collection tubes by adding 1 M of Tris pH 9.0 (0.1 mL per mL of each eluted fraction). Elute with 3 CV elution buffer (0.1 M of glycine-HCl, pH 2.7), and collect the eluted fraction; repeat 3 times.
    5. Dialysis the eluted fractions in 2 L of PBS using dialysis tubing (12.4 kDa) at 4 °C for 2 h. Repeat twice.
    6. Determine the protein concentration by ultramicrospectrophotometer (Molar extinction coefficient: 91105. 1 A280 = 0.69 mg/L).

4. SDS-PAGE and Western-blotting Analysis

  1. Take a 10 µL of the sample into 5x SDS loading buffer and mix well. Boil the protein mixture at 100 °C for 5 min.
    1. Prepare 5x SDS loading buffer: 250 mM of Tris-HCl, pH 6.8, 10% wt/vol SDS, 0.5% wt/vol bromophenol blue, 50% vol/vol glycerol, 5% vol/vol beta-mercaptoethanol.
  2. Prepare an SDS-PAGE gel with a loading gel (5%) and separation gel (12%) as previously described26,27,28.
  3. Load 10 µL of the boiled protein mixture and protein marker, and run the SDS-PAGE.
  4. Stain the gel by shaking for 20 min with Coomassie brilliant blue solution, and then perform destaining.
  5. For western blotting, after transferring to a PVDF membrane, block the membrane with TBST (20 mM of Tris-HCl, 150 mM of NaCl, 0.1% vol/vol Tween 20) + 5% wt/vol skim milk for 1 h at room temperature while shaking.
  6. Incubate the membrane with primary antibodies (anti-His or anti-Flag) diluted 1:2,000 in TBST + 5% skim milk for 1 h.
  7. Wash with TBST for 5 min at room temperature, and repeat 3 times.
  8. Incubate the membrane in secondary antibody (HRP-linked anti-mouse Fc antibody) diluted 1:2,000 in TBST + 5% skim milk for 1 h.
  9. Wash with TBST for 5 min at room temperature, and repeat 3 times.
  10. Add 1 mL of HPR substrate per film, develop and take images.

5. Gel Filtration Analysis

  1. Use the following column: Column volume of 24 mL; Equilibration buffer of PBS pH 7.4; Flow rate of 0.5 mL/min at room temperate; Sample volume of 200 µL.
  2. Use the following protein volume and concentration: 500 µL of 1.2 mg/mL GPC3-S-Fab. Centrifuge at 20,000 x g for 30 min, and take 200 µL to load.
  3. For the protein marker volume and concentration, take 100 µL of cytochrome C (2 mg/mL, 12.4 kDa), 140 µL of anhydrase (3 mg/mL, 29 kDa), 140 µL of albumin (10 mg/mL, 66 kDa) and 200 µL of alcohol dehydrogenase (5 mg/mL, 150 kDa) into one tube. Centrifuge at 20,000 x g for 30 min, and take 200 µL to load.
  4. Before each run, wash with at least 2 CV of distilled water at a flow rate of 0.5 mL/min.
  5. Equilibrate the column with at least 2 CV of equilibration buffer (PBS, pH 7.4) at a flow rate of 0.5 mL/min.
  6. Automatically inject the sample at 0.5 mL/min to load the sample.
  7. Elute with equilibration buffer at 0.5 mL/min and detect at 280 and 215 nm.

6. Flow Cytometry Analysis

  1. Culture the HepG2 (GPC3 positive), Hep3B (GPC3 positive), Huh7 (GPC3 positive), and MHCC-97H (GPC3 negative) cells in 100 mm x 20 mm plastic dishes containing DMEM medium with 10% fetal bovine serum (FBS), penicillin (100 µg/mL), and streptomycin (50 µg/mL) at 37 °C, 5% CO2.
  2. Culture CHO (GPC3 negative) cells and CHO/GPC3 cells harboring the full-length human GPC3 cDNA (GPC3 positive) in 100 mm x 20 mm plastic dishes containing RPMI 1640 medium with 10% FBS, penicillin (100 µg/mL), and streptomycin (50 µg/mL) at 37 °C, 5% CO2.
  3. Culture the NK92/CD16 harboring the full-length human CD16 cDNA (CD16 positive) in 25cm2 flask containing RPMI 1640 medium with 10% FBS, penicillin (100 µg/mL), and streptomycin (50 µg/mL) at 37 °C, 5% CO2.
  4. When the cells are 70 - 90% confluent, discard the medium and wash the cells with 2 mL of PBS. Add 1 mL of 0.25% trypsin and incubate at 37 °C for 2 - 3 min (or until the cells start to detach from the surface). Add 1 mL of the complete growth medium to neutralize the trypsin and re-suspend the cells by gently pipetting up and down.
  5. Count the cell numbers using a Neubauer hemocytometer and detect the cell viability by Trypan Blue.
  6. Collect 3 x 106 cells for each cell line. Add 1 mL of PBS + 0.2% BSA to re-suspend the cells. Centrifuge at 200 x g, 4 °C for 5 min. Repeat twice.
  7. Add 0.3 mL of PBS + 0.2% BSA to re-suspend the cells, and transfer 100 µL (approximately 1 million) to each 5 mL polystyrene round-bottom tube.
    NOTE: Please make sure all steps from here to the tubes should be kept cold on ice. When the antibody is binding to the cell surface antigen, the complex will start to internalize. By keeping it cold, the process is slowed significantly.
  8. Add 10 µL of primary antibodies (GPC3-S-Fab or human IgG1, final concentration 5 µg/mL) to every tube, and incubate on ice for 1 h.
  9. Add 1 mL of PBS + 0.2% BSA to wash, and centrifuge at 200 x g, 4 °C for 5 min. Repeat three times.
  10. Re-suspend in 100 µL of PBS + 0.2% BSA, and then add 0.5 µL of secondary antibody (anti-human IgG(H+L)-488, final concentration 10 µg/mL) and incubate on ice for 1 h. NOTE: From this step, please keep the cells from the light.
  11. Add 1 mL of PBS + 0.2% BSA to wash, and centrifuge at 200 x g, 4 °C for 5 min. Repeat three times.
  12. Analyze the cells by flow cytometer according to the standard protocol29. Acquire cells with a 488 nm laser for excitation.

7. Cytotoxic Assays

  1. Prepare tumor cells.
    1. Culture HepG2, Hep3B, Huh7, MHCC-97H, and CHO cell lines as described in steps 6.1 - 6.5.
    2. Dilute cells to 0.5 × 105/mL, and plate 100 µL cells (5,000 cells per well) on 96-well plates. Culture 6 - 8 h to allow the cells to attach.
  2. Prepare human peripheral blood mononuclear cells (PBMCs).
    1. Dilute fresh prepared blood with an equal volume of PBS in a 50 mL conical tube. For example, dilute 10 mL of blood with 10 mL of PBS.
    2. Take 15 mL of lymphocyte separation medium into a fresh 50 mL conical tube. Transfer the diluted blood onto the lymphocyte separation medium along the tube side slowly.
      NOTE: Keep the tube vertical. Do not break the surface of the lymphocyte separation medium.
    3. Centrifuge at 400 x g for 35 min at room temperature with the brake off.
    4. Slowly remove the upper plasma layer, and take the white blood layer containing PBMCs at the plasma-lymphocyte separation medium interface.
    5. Dilute the PBMCs with a double volume of PBS + 2% FBS. Centrifuge at 400 x g for 5 min.
    6. Wash the cells twice with PBS + 2% FBS. Centrifuge at 400 x g for 5 min. Count the cell numbers as described in step 6.5.
  3. Prepare NK cells.
    1. Prepare the PBMCs suspension at a concentration of 5 x 107 cells/mL in PBS + 2% FBS, and place them in a 5 mL polystyrene round-bottom tube.
    2. Add Human NK cell Enrichment Cocktail at 50 µL/mL of cells. Mix well, and incubate at room temperature for 10 min.
    3. Resuspend the Magnetic Particles from the NK cell Enrichment kit, and mix well to ensure they are uniformly suspended by pipetting up and down vigorously more than 5 times. Transfer the resuspended Magnetic Particles at 100 µL/mL to the cell suspension. Mix well, and incubate at room temperature for 5 min.
    4. Bring the cell suspension up to a total volume of 2.5 mL by adding PBS + 2% FBS. Mix the cells in the tube by gently pipetting up and down 2 - 3 times. Place the tube (without a cap) into the magnet. Let the magnetic beads settle down for 2.5 min at room temperature.
    5. Remove the magnet. In one continuous motion, invert the tube, pouring off the enriched cell suspension into a new tube.
    6. Count the cell numbers as described in step 6.5.
  4. Set up the cytotoxic reaction.
    1. Dilute the NK cells to 5 × 105 cells/mL using the corresponding culture medium. Add 100 µL of NK cell suspension to the tumor cells plated on a 96-well plate.
    2. Add 10 µL of GPC3-S-Fab at different concentrations, with the highest dose at a final concentration of 30 µg/mL, 3-fold dilution, 9 point (three replicates).
    3. Incubate at 37 °C, 5% CO2 for 72 h.
    4. After 72 h of incubation, remove the medium, and add 100 µL of DMEM medium containing 10 µL of CCK8 reagent to each well in 96-well plates, and then incubate at 37 °C.
    5. Read the plate at 450 nm at 1, 2, 3, and 4 h after adding the CCK8 reagent.
    6. Analyze the data. Calculate the survival rate as (OD450 GPC3-S-Fab + Effector + tumor - OD450 medium)/(OD450 tumor - OD450 medium) × 100% and (OD450 GPC3-S-Fab + tumor - OD450 medium)/(OD450 tumor - OD450 medium) × 100%. The effector cells are NK cells.

Results

GPC3-S-Fab purification

GPC3-S-Fab was purified from E. coli by a two-step affinity purification, first with Ni-NTA-agarose, followed by IgG-CH1 affinity purification. After the two-step affinity purification, GPC3-S-Fab was purified to homogeneity with the two chains close to 1:1 (Figure 2A). The presence of both VH-CH1-CD16 VHH and VL-CL polypeptides can be identified by ...

Discussion

In this study, we present a strategy to construct a new format of bsAbs, GPC3-S-Fab, which can recruit NK cells targeting GPC3 positive tumor cells. The S-Fab is based on the natural Fab format by adding an anti-CD16 VHH11,12. Compared with the bsAbs containing Fc region, GPC3-S-Fab can easily be produced in the periplasm of bacteria on a large scale.

Using the expression and purification strategy described in the protocol, we obtained...

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This work was financially supported by the R&D Plan of Guangdong Province (PR China) (2016A050503028).

Materials

NameCompanyCatalog NumberComments
Shaking incubatorThermo FisherMAXQ 4000
Shaking incubatorZhichengZWYR-D2402
CentrifugeCenceGL-10MD
CentrifugeBeckman coulterAvanti j-26S XPI
Centrifugeeppendorf5810R
Ultraviolet spectrophotometerThermo FisherNanodrop
Analytical polyacrylamide gel electrophoresis apparatusMini-PROTEAN® TetraBio-rad
Trans-blot apparatusCriterionBio-rad
Imaging systemBio-radchemidoc tm XRS+
Fast Protein Liquid chromatogramGE HealthcareAKTA avant
GF columnGE Healthcare28-9909-44 Superdex 200 Increase 10/300 GL
Flow CytometerBeckman coulterFC500
Centrifugeeppendorf5702R
Envision plate readerTECANInfinite F50
Anti His-tageBioscience14-6657-82
anti-Flag-tagSigmaF1804
anti-human(H&L)-488A11013Invitrogen
Anti-mouse IgG HRP-linked antibodyCell Signaling7076S
Ni-NTA-AgaroseTribioscienceTBS9202-100
IgG-CH1 affinity resinThermo Fisher194320005
Ficoll-Plaque PlusGE Healthcare17-1440-03
NK cell enrichment kitStemcell19055
MagnetStemcell18000
CCK8 kitDojindoCK04
DMEMGibcoC11995500CP
RPMI-1640GibcoC11875500CP
Fetal Bovine Serum (FBS)SigmaF2442
TrypsinGibco15050-057
Penicillin-StreptomycinGibco15140-122Cell culture
Standard markerSigma AldrichMWGF200Gel filtration
Isopropyl-b-D-thio-galactopyranoside (IPTG)VWR chemicalsVWRC0487-100G
Dialysis tubingSigma AldrichD0655-100FT
KnanamycineVWRVWRC0408-100G
AmpicillinVWRVWRC0339-100G
TryptoneThermo FisherLP0042B
Yeast ExtractThermo FisherLP0021B
NaClSangon BiotechA100241
Trizma baseSigma AldrichT6791-1KG
EDTASigma AldrichV900106
GlycinealaddinA110752-500g
KClaladdinP112134-500g
MgClSigma AldrichV900020
AgarSangon BiotechA505255-0250
Potassium Phosphate, Monobasic Anhydrous (KH2PO4)VWR7778-77-0
Sodium Phosphate, Dibasic, Anhydrous (Na2HPO4)VWR7558-79-4
2-MercaptoethanolVWR60-24-2
Phenylmethyl Sulfonyl Fluoride (PMSF)VWR329-98-6
LysozymeSigma AldrichL6876-25G
Coomassie Brilliant Blue R250VWRVWRC0472-50G
Bromophenol blueSangon BiotechA500922-25G
Bovine Serum Albumin (BSA)VWRVWRC0332-100G
GlycerolSigma AldrichV900122
100mm x 20mm plastic dishCorning430167
25cm2 flaskCorning430639
96 well cell culture clusterCorning3599
SucroseSangon BiotechA610498-0005
CHOthe Type Culture Collection of the Chinese Academy of SciencesGNHa 3
MHCC-97Hthe Type Culture Collection of the Chinese Academy of SciencesSCSP-528
HepG2the Type Culture Collection of the Chinese Academy of SciencesTCHu 72
Huh7the Type Culture Collection of the Chinese Academy of SciencesTCHu182
Hep3Bthe Type Culture Collection of the Chinese Academy of SciencesTCHu106
NK92ATCCCRL2408

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