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May 2021

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Protocol for High Throughput Screening of Antibody Phage Libraries
抗体噬菌体文库的高通量筛选方案   

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Abstract

Phage display is a proven and widely used technology for selecting specific antibodies against desired targets. However, an immense amount of effort is required to identify and screen the desired positive clones from large and diverse combinatorial libraries. On the other hand, the selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias toward clones with randomly produced amber stop codons, making it more difficult to identify desirable binding antibodies. To overcome the screening of desired clones with amber codons, we present a step-by-step approach for effective phage library screening to isolate useful antibodies. The procedure calls for creating a simple new vector system for soluble production of phage ELISA positive binding clones with one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences, which is otherwise difficult in standard screening.


Graphical abstract:


Keywords: Phage display (噬菌体展示), Amber codon (琥珀密码子), scFv (scFv), High-throughput screening (高通量筛选), Novel vector system (新型矢量系统 )

Background

Biomolecules based on monoclonal antibodies are commonly utilized for disease detection and prevention (Borghardt et al., 2018; Parray et al., 2020; Kumaret al., 2022). The single-chain variable fragment (scFv) antibody is one of the most often exploited biomolecules because it is the smallest antibody unit and has low immunogenicity and low-cost production properties (Kumar et al., 2019b; Parray et al., 2020). The scFv is the most commonly employed combinatorial therapeutic entity, either alone or in combination with other medications (Frenzel et al., 2016; Kumar et al., 2019b). An antibody in the form of scFv has variable heavy (VH) and light-chain (VL) sections that are linked by an efficient linker that can be effectively produced in E. coli (Kumar et al., 2012; Kumaret al., 2019a). The phage display technique is the most popular and successful way of generating scFv antibody fragments among all in vitro display methods. The size and functional diversity of the library used for screening enhances the efficiency of isolating scFv molecules from phage display antibody libraries. The most common issue with the soluble expression of scFv clones from phage libraries is the higher frequency of amber codons within the scFv gene, resulting in the premature expression of scFv clones in non-suppressor E. coli strains. This is more common in the case of synthetic and semi-synthetic libraries because these libraries are constructed randomly at few residues—particularly at NNN, NNK, NWG, NWC, and NSG codons—which increase the biased inclusion of amber codons (Marcus et al., 2006). Due to the frequent presence of amber codons within antibody gene sequences, the isolation of functional soluble scFv molecules is the most prevalent problem encountered during the screening of synthetic and semi-synthetic libraries, resulting in premature expression of scFv clones in non-suppressor E. coli strains (Barderas et al., 2006; Perween et al., 2021a). However, the inclusion of an amber stop codon does not affect the display of scFvs on the phage surface in E. coli suppressor strains, but it reduces the overall yield in terms of the total number of functional soluble scFv protein-expressing clones.


Directing individual scFv genes to be resynthesized or using Kunkel mutagenesis are two popular ways to solve this problem. Both of these processes become expensive and time demanding, considering when the purpose is to screen a substantial proportion of clones, especially in viral targets where a large amount of screening is essential to generate a small number of neutralizing clones (Reader et al., 2019).


In this Bio-protocol, we describe a novel strategy for rapid screening of scFvs containing amber codons and turning them into usable soluble scFvs that can be applied to several phage antibody libraries. We discuss a fast and reliable screening strategy that can be used to screen a large number of phage antibody libraries with amber stop codons (TAG) in the encoding series.

Materials and Reagents

All chemicals are of Analytical Reagent Molecular Biology/Tissue culture grade.

PRODUCT NAME CATALOGUE NUMBER COMPANY NAME
(3-(N-morpholino) propane sulphonic acid) MOPs M1254 Sigma-Aldrich
2× YT media G034-500G Himedia
Absolute ethanol 24102 Sigma-Aldrich
Acetic acid W200603-1KG-K Sigma-Aldrich
Acrylamide A8887-100G Sigma-Aldrich
Agarose MB080-100G Himedia
Alkaline Phosphatase Blue Membrane substrate solution

AB0300

Sigma-Aldrich
Ampicillin SD002 Himedia
Anti-rabbit HRP Code: 111-035-144 Jackson Immune Research
Beta-mercapto ethanol 21985023 ThermoFisher
Bis-Acrylamide A2792-100ml Sigma-Aldrich
Boric acid MB007 Himedia
Bovine Serum Albumin (BSA) A3059-10G Sigma-Aldrich
Bright-Glo Luciferase assay system E2610 Promega
Bromophenol Blue B0126-25G Sigma-Aldrich
Calcium chloride GRM710 Himedia
Cut smart buffer B6004S New England Biolabs
Cyclosporine RM8155 Himedia
DEAE-dextran MB145 Himedia
Diethanolamine RM8218 Himedia
Diethyl pyro carbonate (DEPC) D43060 RPI – Research Products International
Dimethyl sulphoxide (DMSO) 673439 Sigma-Aldrich
DpnI enzyme R0176S New England Biolabs
Dulbecco’s Modified Eagle Medium (DMEM) 11965118 GibcoTM
Ethylene diamine tetra acetic acid GRM678 Himedia
EXpi 293F cells 100044202 ThermoFisher
Ficoll 26873-85-8 Sigma-Aldrich
Gel extraction kit 28706X4 Qiagen
Gelatin G2500 Sigma-Aldrich
Glucose MB037 Himedia
Glycerol MB060 Himedia
Glycine MB013 Himedia
HisPurTM Ni-NTA Magnetic Beads 88832 Thermo ScientificTM
Histopaque 10771 Sigma-Aldrich
Hydrocortisone RM556 Himedia
Hydrogen chloride 18-603-211 ThermoFisher
Imidazole MB019-100G Himedia
Isopropyl β-D-thiogalactoside RM2578 Himedia
Kanamycin Sulphate MB105 Himedia
L-glutamine 25030081 GibcoTM
Ligase 15224017 InvitrogenTM
Ligase buffer 46300018 InvitrogenTM
LMB3 primer Custom DNA oligos Integrated DNA Technologies IDT
Luria Broth M1245 Himedia
Luria Broth Agar M1151-500G Himedia
Magnesium chloride MB237 Himedia
Magnesium sulphate GRM1281 Himedia
Methanol 322415-250ml Sigma-Aldrich
Mini prep kit 27106X4 Qiagen
Mono Sodium Phosphate GRM3964 Himedia
NcoI-HF® R3193S New England Biolabs
Ni NTA beads 88221 ThermoFisher
NotI-HF® R3189S New England Biolabs
Penicillin SD028 Himedia
PHEN primer Custom DNA oligos Integrated DNA Technologies IDT
Phosphate buffered saline TS1101-20L Himedia
Phytohemagglutinin (PHA) 10576015 GibcoTM
PierceTM Protein G Magnetic Beads 88848 Thermo ScientificTM
Polybrene (Hexadimethrine bromide) H9268 Sigma-Aldrich
Polyethylene glycol MB149-500G Himedia
Potassium Acetate W292001 Sigma-Aldrich
Potassium chloride P3911-25G Sigma-Aldrich
Potassium dihydrogen phosphate PO662-25G Sigma-Aldrich
RNase A EN0531 ThermoFisher
RPMI-1640 medium 11875093 GibcoTM
Skim milk GRM1254 Himedia
SnakeSkinTM Dialysis Tubing 88244 Thermo ScientificTM
Sodium Azide GRM1038 Himedia
Sodium bicarbonate GRM849 Himedia
Sodium carbonate GRM851 Himedia
Sodium chloride MB023-1KG Himedia
Sodium dodecyl-sulphate 0227-10G VWR Life science
Sodium hydroxide 72064 Sigma-Aldrich
Sodium phosphate dibasic Bio Reagent NIST2186II Sigma-Aldrich
Stop solution N600 Thermo Fisher Scientific
Streptomycin Sulphate CMS220 Himedia
Sucrose MB025 Himedia
TG1 Electrocompetent Cells 23227 Lucigen
TMB (Tetramethylbenzidine) Substrate solution N301 Thermo Fisher Scientific
Tris base TC072 Himedia
Tris Buffered Saline R017R.0000 ThermoFisher
Tris free base MB029-500G Himedia
Tris-HCl MB030 Himedia
Triton X100 MB031 Himedia
Trypan blue T8154 Sigma-Aldrich
Trypsin TC598 Himedia
Tween 20 MB067 Himedia

  1. Goat affinity purified Antibody to human IgG Fc, alkaline phosphatase conjugated goat affinity purified antibody to IgG Fc, and purified human IgG whole molecule were purchased from Cappel, MP Bio, USA.

  2. A human mAb 1418 against parvovirus B19 was gifted by Dr. Zolla Pazner, NYU SoM, USA


Plasticware

All plasticware used is disposable (glassware is not used in this work).

NAME CATALOGUE NUMBER COMPANY NAME
96 well flat bottom Immunol plate for ELISA CLS3370 Corning
96 well round bottom Immunol plate for ELISA CLS3367 Corning
96 well tissue culture plate CLS 3628 Corning
Disposable pipettes of 5 mL, 10 mL and 20 mL CLS4487 Corning
Microfuge tubes 1.5 mL CLS3620 Corning
PCR tubes of 0.2 mL PCR-02-A Axygen
Petri dishes 460062 Tarson
Pipette tips 0.5–10 μL AXYT300RS Corning
Pipette tips 1–200 μL CLS4860 Corning
T-25 cm3 tissue culture flask C6231 Corning
T-75 cm3 tissue culture flask C7231 Corning


BUFFERS

  1. 10× stock of gel loading dye (see Recipes)

  2. 2× Sample buffer (see Recipes)

  3. Acrylamide:Bis (100 mL) (see Recipes)

  4. Antibiotic concentration (see Recipes)

  5. Buffer for Agarose Gel Electrophoresis (see Recipes)

  6. Coating Buffer (see Recipes)

  7. Composition of Reagents (see Recipes)

  8. Counting of Expi293FTM Human Cells (see Recipes)

  9. Destaining solution I (see Recipes)

  10. Elution Buffer (1 L) (see Recipes)

  11. Lower Gel Buffer [pH 8.8] 200 mL (see Recipes)

  12. Lysis buffer (1 L) (see Recipes)

  13. Phosphate buffered saline (see Recipes)

  14. Purification of scFvs (see Recipes)

  15. Tank Buffer 1× 2L (pH 8.3) (see Recipes)

  16. Upper Gel Buffer [pH 6.8] 100 mL (see Recipes)

  17. Wash Buffer (1 L) (see Recipes)

  18. Wash Buffer (see Recipes)

  19. Western Blotting solution (see Recipes)

Procedure

  1. HELPER PHAGE PRODUCTION

    A helper phage is necessary for transferring phagemid particles into E. coli. Phagemid particles contain (i) an antibiotic maker, (ii) antibody-G3P fusion protein, and (iii) phage origin of replication.

    The phagemid libraries are amplified along with the antibody-G3P fusion protein and helper phage genes, which are required for infection, replication, assembly, and budding.

    1. Take three Corning® 50 mL Falcon centrifuge tubes (FCTs) and label them A, B, and C.

    2. Add 5 mL of 2× YT media to each FCT tube.

    3. Take B and C as negative controls by adding ampicillin to one and kanamycin to the other.

    4. Add 20 μL of TG1 E. coli cells to all three FCTs.

    5. Incubate the FCTs at 37°C with shaking for overnight growth.

    6. Subculture 20 μL of the previously inoculated TG1 cells in FCT A (Step A1) in 5 mL of fresh 2× YT media; incubate for 2–4 h at 37°C with shaking.

    7. Then, add 40 μL of helper phage to the cultured TG1 cells.

    8. Grow for 30 min at 37°C without shaking.

    9. Grow for 30 min at 37°C with shaking.

    10. Add this culture to 200 mL of fresh 2× YT media with kanamycin in a 50 μg/mL working concentration.

    11. Allow to grow overnight at 30°C with shaking.

    12. Remove the flask from the incubator, collect the culture in an autoclaved caesium bottle.

    13. Centrifuge at 14,260 × g for 30 min at 4°C.

    14. Pour the supernatant into another fresh caesium bottle and centrifuge at 14,260 × g and 4°C for 30 min.

    15. Without disturbing the pellet, collect the supernatant in a glass bottle and add PEG/NaCl [20% (wt/vol) Polyethylene glycol 6000, 2.5 M NaCl], keeping the ratio of the supernatant and PEG/NaCl as 50:15.

    16. Store in a cold room at 4°C for 4–5 h, or overnight for better results.

    17. Spin the culture at 14,260 × g and 4°C for 1 h to allow the cells to settle down.

    18. Without disturbing the pellet, discard the supernatant, and resuspend the pellet with 1× PBS, keeping it in 1.5 mL tubes.

    19. Centrifuge the microtubes at 16,200 × g for 5 min. In case a pellet is formed, transfer the supernatant into fresh tubes and store at 4°C ( Figure 1 ).



    Figure 1. Schematic representation of the steps involved in Helper Phage preparation.


  2. GROWING TOMLINSON I + J AND MAKING SECONDARY STOCK

    1. Take 100 mL of 2× YT media containing ampicillin and 1% (vol/vol) glucose. To this media, add 500 μL of the Tomlinson I + J phage library stock.

    2. Incubate for 1–2 h at 37°C with shaking, until the O.D. at 600 nm is approximately 0.4.

    3. Divide the 100 mL of culture media into two parts. First, use 50 mL to grow the library; then, use the remaining media to make secondary stocks of the library.


    Growing the library (phage stocks):

    1. Take 50 mL of the 100 mL of grown media and add 200 μL of helper phage.

    2. Incubate for 30 min at 37°C without shaking.

    3. Centrifuge at 1,200 × g for 10 min.

    4. Carefully discard the supernatant without disturbing the pellet.

    5. Dissolve the pellet in 100 mL of 2× YT media containing 100 μg/mL ampicillin, 50 μg/mL kanamycin, and 0.1% (wt/vol) glucose.

    6. Incubate the resuspended pellet overnight at 30°C with shaking.

    7. After overnight incubation, transfer the culture to a centrifuge bottle (caesium bottle) and centrifuge at 14,260 × g and 4°C for 30 min.

    8. Transfer the supernatant to another caesium bottle and centrifuge again at 14,260 × g and 4°C for 30 min.

    9. Carefully transfer the supernatant into another autoclaved glass bottle and discard the pellet. Add PEG/NaCl (20% Polyethylene glycol 6000, 2.5 M NaCl) to the supernatant collected (15 mL of PEG/NaCl to 50 mL supernatant).

    10. Store this in a cold room at 4°C for 4–5 h, or overnight for better results.

    11. Spin the culture at 14,260 × g and 4°C for 1 h to allow the cells to settle down.

    12. Without disturbing the pellet, discard the supernatant and resuspend the pellet with 1× PBS; keep it in 1.5 mL tubes.

    13. Centrifuge the microtubes at 16,200 × g for 10 min. In case a pellet is formed, transfer the supernatant into fresh tubes and store at 4°C for short term storage. Add 15% (vol/vol) glycerol for longer storage at -80°C ( Figure 2 ).


    Making secondary stocks of phage library:

    1. Grow the remaining 50 mL of media further for 2 h at 37°C with shaking.

    2. Allow the cells to settle down by centrifuging the culture at 1,500 × g for 15 min.

    3. Resuspend the pellet in 3 mL of 2× YT media containing 15% (vol/vol) glycerol.

    4. Store at -80°C until further use ( Figure 2 ).



    Figure 2. Schematic representation of the steps involved in library amplification for screening purposes and library secondary stock preparation.


  3. BIO-PANNING

    In this step, the target proteins are immobilized onto the surface of the microtiter plate. The first step is the addition of the rescued Tomlinson phage library. The second step involves binding, where the phage displaying scFvs, the highest affinity antibodies, bind the epitopes of the antigen, and those with low binding affinity are removed by washing. The antigen bound phage are eluted by enzymatic digestion using trypsin. The eluted phage are infected to TG1 followed by addition of helper phage for amplification. To accumulate phage displaying high-affinity antibody fragments, these steps were repeated three times with the amplified phage from the preceding round of panning, and each time, the number of washing cycles is increased.


    ROUND 1

    1. One day before the experiment, coat one row (say row C) of the ELISA plate, namely plate A, with the required antigen (100 μL per well), with a concentration of 5 μg. Coat the antigen with coating buffer.

    2. Incubate plate A at 4°C overnight.

    3. Next day, make 3% BSA in 5 mL of PBS and incubate for 10 min at 37°C. Then, coat another plate, i.e. , plate B; coat two rows (say rows C and D) and incubate the plate at 37°C for 1 h.

    4. Plate C: coat one row (say row C) with a pinch of skim milk in 1 mL of autoclaved PBS + 700 μL of phage stock. Coat 100 μL per well.

    5. Incubate plate C at room temperature for 30 min. Then, transfer the coating from row C to any other row, say row D. Incubate for 30 min more.

    6. After 1 h of coating plate B, wash the plate once with autoclaved PBS (250 μL per well) and transfer the content of plate C onto row C of plate B.

    7. Again, incubate plate B at room temperature for 30 min and then transfer the content of row C onto row D, followed by another 30 min incubation.

    8. While washing plate B, simultaneously wash plate A, by using autoclaved PBS (250 μL per well) and then block with 3% (wt/vol) BSA (200 μL per well). Incubate at room temperature for 1 h.

    9. Wash plate A three times with autoclaved PBS.

    10. Then, transfer plate B content onto plate A; followed by incubation for 1 h at room temperature.

    11. After incubation, wash plate A with PBST 10 times. Make 1 mL of PBS containing 50 μL of trypsin and add 95 μL of this solution to each well.

    12. Keep the plate for 10 min at 37°C.

    13. Then, collect all the trypsinized content into one aliquot. This will be the output of bio-panning round 1.

    14. Use the output of bio-panning 1 to calculate the transducing unit (TU) ( Figure 3 ).



      Figure 3. Schematic representation of the steps involved in the bio-panning process.


    Preparation of the next round of bio-panning

    1. Add 500 μL of bio-panning output into 5 mL of TG1 cell growth media.

    2. Keep for 30 min at 37°C with shaking.

    3. Centrifuge at 700 × g for 10 min.

    4. Use 1 mL of supernatant to dissolve the pellet formed during centrifugation and throw the rest of the supernatant out.

    5. Spread this on a bioassay dish containing 2× YT agar with ampicillin.

    6. Allow the bacteria to grow at 37°C overnight.

    7. Next day, add 3–5 mL of 2× YT media containing 15% glycerol onto the bioassay dish and scrape all the colonies grown overnight. Collect them in a fresh tube.

    8. Use 100 μL of the scraped colonies and store the rest.

    9. Add 100 μL of scraped colonies to 50 mL of 2× YT media containing ampicillin (100 µg/mL) and 1% (vol/vol) glucose.

    10. Allow it to grow for 2 h at 37°C with shaking.

    11. Take 10 mL of the above culture and add a 40 μL of helper phage. Incubate for 30 min at 37°C without shaking.

    12. Centrifuge the culture at 700 × g for 15 min and discard the supernatant without disturbing the pellet.

    13. Dissolve the pellet in 50 mL of 2× YT media containing 100 µg/mL ampicillin, 50 µg/mL kanamycin, and 0.1% glucose. Incubate overnight at 30°C with shaking.

    14. Centrifuge the overnight grown culture at 14,260 × g and 4°C for 30 min, collect the supernatant in a fresh cesium bottle and centrifuge again at 14,260 × g and 4°C for 30 min.

    15. Carefully transfer the supernatant into an autoclaved glass bottle and discard the pellet. Add PEG/NaCl (20% Polyethylene glycol 6000, 2.5 M NaCl) to the supernatant collected (15 mL of PEG/NaCl to 50 mL of supernatant).

    16. Store this in a cold room at 4°C for 4–5 h, or overnight for better results.

    17. Spin the culture at 14,260 × g for 1 h at 4°C to allow the cells to settle down.

    18. Without disturbing the pellet, discard the supernatant and resuspend the pellet with 1× PBS, keeping it in 1.5 mL tubes.

    19. Centrifuge the microtubes at 16,200 × g for 10 min. In case a pellet is formed, transfer the supernatant into fresh tubes and store it at 4°C.

    20. The collected phage is to be used as an input in the next round of bio-panning by coating this on plate C instead of phage stock.

    Note: With each bio-panning round, decrease the antigen coating concentration (for example, round 1 with 5 μg/μL, round 2 with 3 μg/μL, and round 3 with 1.5 μg/μL) and calculate the TU of every input and output used during phage selection.


  4. SCREENING BY PHAGE ELISA

    Day 0: One day before performing ELISA

    1. For the phage selection process, grow the colonies of the last bio-panning round output. Inoculate the colonies formed during the last round of bio-panning output; each inoculation is done in 5 mL of 2× YT media containing 100 μg/mL ampicillin.

    2. Incubate for 30 min at 37°C without shaking. Wait until OD 600 of the culture reaches 0.4–0.6.

      Back up set: At this step, take 200 μL of the culture of each clone and add 200 μL of autoclaved 50% (vol/vol) glycerol solution to make a stock and store at -80°C for future experiments

    3. Add 20 μL of helper phage.

    4. Incubate again for 30 min at 37°C with shaking.

    5. Add 50 μg/mL of kanamycin and incubate overnight at 30°C with shaking at 140–160 × g .

    6. Coat the 96 well assay plate with the required antigen with a concentration of 2 μg/μL. Do BSA coating as negative control and keep the plate overnight at 4°C ( Figure 4 ).



      Figure 4. Schematic representation of the steps involved in the Phage ELISA screening process.


    ELISA

    1. Next day, take the antigen-coated plate out of 4°C and wash once with PBS.

    2. Block the plate with 200 μL of 5% skim milk in PBS per well.

    3. Incubate the plate for 1 h at room temperature.

    4. Take all the colony inoculation out of the incubator and centrifuge the tubes at 3,900 × g for 20 min.

    5. After 1 h blocking, wash the ELISA plate three times with PBS (250 μL).

      Note: Blocking can be extended to 90 min if required, based on the timing of the parallel steps

    6. After 1 h of incubation, wash the plate three times with PBS.

    7. Then add 100 μL of primary antibody per well, i.e. , 50 μL of supernatant from all the centrifuged tubes and 50 μL of skim milk (diluted in 1:1).

    8. Incubate for 1 h at room temperature.

    9. Wash the plate with 250 μL of 0.1% PBST four times.

    10. Add 100 μL of secondary antibody per well (1:2,000). Follow with a 1 h incubation at room temperature in the dark.

    11. Wash the plate with 250 μL of 0.1% PBST six times.

    12. Add 100 μL of the substrate (TMB) to each well.

    13. Allow the reaction to take place for 15–20 min in the dark.

      To stop the reaction, add 50 μL of stop solution (2 NH2SO4 ) per well, and read the plate at 450 nm on a multimode ELISA reader.


  5. ISOLATION OF PLASMID DNA

    1. Identify the positive binding clones in Phage ELISA (at least four times more than the negative control).

    2. Take two FCTs and label them A and B; add 5 mL of 2× YT media to each FCT.

    3. Take B and add ampicillin (100 µg/mL) and C as negative control by adding kanamycin (50 µg/mL) in it.

    4. In FCTs A and B, inoculate from a glycerol stock that is preserved at -80°C.

    5. Incubate the FCT at 37°C with shaking for overnight growth.

    6. Next morning, check tubes A and B. Tube A culture should be turbid, and in tube B, there should be no growth.

    7. Spin down by centrifuging the culture at 2,820 × g for 15 min.

    8. For plasmid isolation, use the Qiagen Miniprep kit following the manufacturer’s instructions.

    9. Check the quality and concentration of the isolated plasmid DNA using a nanodrop spectrophotometer. The 260/280 ratio of the isolated DNA should be 1.8.

    10. Prepare a 0.8% Agarose gel and check the quality of the isolated plasmid DNA on the gel.

    11. Make a 10 μL aliquot of the plasmid DNA, and use LMB3 and PHEN sequencing primers for sequencing the scFv insert sequence.


    Soluble ELISA
    1. Perform ELISA as described in the phage ELISA section.

    2. Add 100 μL of purified scFv and incubate for 1 h at room temperature.

    3. Wash the plate three times with 0.1% PBST.

    4. Use a 1:1,000 dilution of primary antibody (anti-His tag) in 2% MPBS and incubate at room temperature.

    5. Wash three times with 0.1% PBST.

    6. Use 1:2,000 diluted anti-rabbit-HRP conjugated secondary antibody in 2% MPBS and incubate at room temperature, followed by washing, as mentioned above.

    7. Add 100 µL of TMB substrate and allow the color to develop. Once the color appears, add 8 NH2SO4 to stop the reaction.

    8. Read the plate at 450 nm in ELISA reader.


  6. DILUTION AND PLATING FOR TRANSDUCING UNIT (TU) CALCULATION



    Figure 5. Representative image showing dilution preparation strategy for helper phage/library TU calculation.


    1. The plating of each dilution is done on a different plate containing ampicillin.

    2. Pour the mixture of phage and bacteria on a plate and spread slowly using a spreader. Label each plate with the dilution that it contains.

    3. Incubate the plates at 37°C for overnight growth. Next day, count the colonies for TU calculation ( Figure 5 ).


  7. CALCULATE TRANSDUCING UNIT

    TU = (No. of colony × 1000)/(10 × dilution). For a 10-8  dilution plate, we got nine colonies;

    Then the TU is calculated as: (9 × 1000)/(10 × 10-8 ) = 9 × 1010


  8. PREPARATION OF COMPETENT CELLS

    1. Streak E. coli TG1 cells on an LB plate and allow cells to grow at 37°C overnight.

    2. Inoculate a single colony in 5 mL of LB media and grow overnight at 37°C.

    3. Subculture in 100 mL of LB by inoculating 1 mL of an overnight culture of E. coli , and grow at 37°C on a shaker until the O.D. at 600 nm reaches approximately 0.6.

    4. Cool culture on ice immediately, and harvest cells by centrifugation at 4,200 × g and 4°C for 5 min.

    5. Remove the supernatant carefully; remove any traces of supernatant by inverting the centrifuge tube on paper towels.

    6. Resuspend the bacterial pellet in 10 mL of ice-cold 0.1 M CaCl2 (autoclaved) and incubate on ice for 30 min.

    7. Recover cells by centrifugation as described above, resuspended in 5 mL of 0.1 M CaCl2 , and aliquot 200 μL of cells in microcentrifuge tubes.


  9. VECTOR DESIGN

    A representative strategy for vector design is shown in Figure 6 .

    A set of designed PCR primers to replace TAG amber codon into TAA in the junction of the scFv-pIII junction is used for vector construction.

    Forward Primer: 5’CACATCATCATCACCATCACGGGTAATAAGAACAAAAACTCATCTC3’

    Reverse primer: 5’GAGATGAGTTTTTGTTCTTATTACCCGTGATGGTGATGATGATGTG3’.

    PCR Reaction setup:

    Steps Initial Denaturation Cycling 16× Extension Hold
    Temperature 95°C 95°C 52°C 72°C 72°C 10°C
    Time 2 min 30 s 50 s 4 min 5 min


    The PCR product is digested by Dpn 1 enzyme

    PCR product 20 µL
    Dpn 1 enzyme 1 µL
    Cut smart buffer 2.5 µL

    Incubate the reaction mixture at 37°C for 2 h. Tap the tube in between.


    1. Thaw two vials of competent cells from the -80°C freezer on ice for 5–10 min. DO NOT tap at this step.

    2. Add 5 µL of Dpn 1 digested product into one tube, and keep the other tube as blank or negative control. Incubate the cells on ice for 30 min.

    3. After 30 min, place both the tubes in the floater to heat shock for 60 s (at this step, set water bath at 42°C).

    4. Immediately place the tubes in ice for 5 min; at this step, pre-warm the media at 37°C.

    5. Add 900 µL of pre-warmed 2× YT medium to the cells in the tube.

    6. Incubate the tubes in the shaker for 60 min at 220 rpm to grow the cells.

    7. Take out the tubes from the shaker, aspirate 100 µL of cell suspension, and plate/spread on LB-Agar-ampicillin plates. Incubate plates in a 37°C incubator for 16 h or overnight.

    8. Next morning, take out the plates, count the colonies, and store at 4°C until further use.

    9. Add 5 mL of 2× YT supplemented with a standard concentration of ampicillin to five tubes. Pick a single colony for inoculation and culture of the colony obtained on the plates; incubate at 37°C with 220 rpm shaking. Incubate also one tube of media as a control.

    10. Next morning, isolate the plasmid DNA from all four tubes using the Qiagen plasmid isolation kit as per the manufacturer’s instructions.

    11. Check the quality of the isolated plasmid using a spectrophotometer by observing the 260/280 DNA ratio.

    12. Aliquot the DNA and send these samples for sequencing using vector-specific primers.

    13. Analyze the DNA sequence data for point mutations and mark the positive clones for further use. Discard the negative clones.


    Digestion of designed vector

    Reaction mixture for restriction digestion:

    Component Volume (μL)
    Plasmid DNA (250 ng·μL-1) 10 μL
    10× CutSmart Buffer (NEB) 5 μL
    Nco I-HF (NEB) 2 μL
    Not I-HF (NEB) 2 μL
    Nuclease free water 31 μL

    Incubate the reaction at 37°C for 2 h.


    1. After 2 h, add 10 μL of 5× gel loading dye and run the sample on a 0.8% Agarose gel, at 100 V for approximately 60 min.

    2. Cut and excise the digested vector DNA from the agarose gel using a sharp surgical blade. Place the excised fragment in a 1.5 mL Eppendorf tube. Purify the digested vector from the excised DNA using the Qiagen Gel extraction kit as per the manufacturer’s protocol.

    3. Evaluate the quality of the purified digested vector DNA using the spectrophotometer by calculating the 260/280 ratio. This purified vector is used for future cloning reactions.


    Digestion of scFv clones

    The phage ELISA binding positive clone that showed no binding in soluble ELISA was further used to isolate plasmid DNA from single colonies, as described in this section.

    The digestion reaction is set up as described below:

    Component Volume ( μL)
    Plasmid DNA (250 ng·μL-1 ) 10 μL
    10× CutSmart Buffer (NEB) 5 μL
    Nco I-HF (NEB) 2 μL
    Not I-HF (NEB) 2 μL
    Nuclease free water 31 μL


  1. After 2 h add 10 μL of 5× gel loading dye and run the sample on a 1% Agarose gel, at 100 V for approximately 40–60 min.

  2. Two bands should be observed in the agarose gel, one band size of approximately 4 kb corresponding to vector DNA and another of 800 bp corresponding to scFv DNA.

  3. Excise the agarose gel slice containing the relevant DNA Fragments (scFv insert 800 bp) and remove extra agarose to minimize the gel slice.

  4. Transfer the gel slice into a microcentrifuge tube and purify using the Qiagen gel extraction kit as per the manufacturer's instructions.

  5. Use this purified scFv DNA for cloning into a newly designed vector.


    Cloning of scFv DNA into newly designed vector

    Component Volume ( μL)
    scFv DNA insert 10 μL
    Designed vector 5 μL
    10× Ligase buffer 2 μL
    Ligase 2 μL
    Nuclease free water 31 μL


  1. Incubate the ligation reaction mixture at room temperature for 4–6 h. Alternatively, this can be kept at room temperature overnight.

  2. After 4–6 h incubation, thaw the TG1 competent cells on the ice for 5 min.

  3. Add 5 μL of ligation mixture to one tube and keep the second tube without insert and/or ligation mixture as a negative control.

  4. Heat-shock the cells for 60 s and immediately keep on ice for 5 min.

  5. Add 900 μL of 2× YT medium and incubate the tubes in a shaker incubator at 37°C for 1 h.

  6. After 1 h, centrifuge the tube at 2,400 × g for 5 min. Decant the supernatant and resuspend the pellet in reaming leftover media. Plate this on pre-warmed 2× YT-Agar plates supplemented with ampicillin, or optionally you can use LB-Agar plates with ampicillin.

  7. Incubate the plates in a 37°C incubator overnight or 12–16 h.

  8. Next morning, count the colonies on the reaction plate. NO COLONIES should be there in the control plate; otherwise, repeat the experiment with all new and freshly prepared reagents.

  9. Inoculate the single colony in 5 mL of 2× YT containing a standard concentration of ampicillin in two 50 mL FCTs. Label one tube as reaction and the other as blank, and incubate both tubes in a shaker incubator (200 to 220 rpm) at 37°C overnight.

  10. Transfer a small inoculum (approximately 4 mL) from the overnight primary culture to a 2 L flask (400 mL media) 2× TY containing 100 μg/mL ampicillin and 0.1% glucose. Grow shaking (250 rpm) at 37°C until the OD600 is approximately 0.9 (approximately 3 to 3.5 h).

  11. Once the OD of the culture reaches 0.9, add isopropyl β-D-thiogalactoside at a final concentration of 1 mM IPTG. Continue shaking (250 rpm) at 30°C overnight.

  12. Harvest the cells by centrifugation at 4,000 × g for 15 min. Store the cell pellet at -20°C if desired or process immediately.



Figure 6. Schematic representation of the modified vector design strategy.

Diagram depicting a modified method for soluble expression of scFv genes with amber stop codons. The amber stop codon (TAG) between the scFv-pIII gene in the original vector is altered to TAA, which prohibits the creation of scFv-pIII fusion proteins. The scFv genes are cloned directly into the modified vector using the same restriction site Nco1/Not1 that is used to clone the scFv gene into the original vector (Perween et al. , 2021a).


Resuspension of cell pellet and cell extract preparation

  1. Adjust pH to 8.0 using NaOH.

  2. Resuspend the pellet in 30 mM Tris-HCl, 20% (wt/vol) sucrose, pH 8.0, at 80 mL/gram of wet weight. Incubate on ice and add 500 mM EDTA dropwise to a final concentration of 1 mM; then incubate the cells on ice for 20 min with gentle agitation.

  3. Clarify the cell suspension by centrifuging at 8,000 × g and 4°C for 20 min.

  4. Collect the supernatant and resuspend the cells in the same volume of ice-cold 5 mM MgSO4 and incubate on ice for 20 min with gentle agitation.

  5. Centrifuge the cells at 8,000 × g and 4°C for 20 min. Collect the supernatant (supernatant is osmotic shock fluid containing periplasmic proteins) and dialyze extensively against lysis buffer.

  6. Filter the dialyzed supernatant through a 0.2 μm filter before continuing with the purification. Equilibrate the resin with lysis buffer (50 mM NaH2PO4 , 300 mM NaCl, 10 mM Imidazole, and pH = 8.0) prior the use of the Ni++ ions.


Purification OfscFvs
  1. For purification, use Ni-NTA beads; add 5 mL of 50% slurry of Ni-NTA-Agarose resin in a purification column and allow the beads to settle down.

  2. Wash the beads using MilliQ (MQ) water; add 10 column volume (CV) of MQ.

  3. Use 10 CV of binding buffer (20 mM Tris; pH = 7.2, 500 mM NaCl and 10 mM Imidazole) to equilibrate the column. Thus, the same pH and buffer composition as that of the Ni++ ion resin ensuring that the sample binds properly

  4. Add the filtered supernatant into the column and allow it to pass through gravitational force.

  5. After the supernatant has been passed, wash the column to remove any impurity. Add 20–30 CV of washing buffer (20 mM Tris; pH = 7.2, 500 mM NaCl, and 25 mM Imidazole).

  6. Elute the bound protein from beads, and use 5–6 CV of elution buffer (20 mM Tris pH = 7.2, 500 mM NaCl, and 500 mM Imidazole). Analyze the protein quality in terms of purity on a 15% Tri-glycine SDS-PAGE.

  7. Dialyze the protein using activated dialysis tubing, clip both the ends of the tube tightly, and put it in a beaker containing cold PBS. With the help of a magnetic stirrer, allow it to spin overnight at 4°C in a cold room.

  8. Mount the Superdex75 Increase column in AKTA FPLC system. Wash the pump thoroughly with PBS.

  9. Use 2 CV of PBS to equilibrate the column; load approximately 500 μL of the purified concentrated protein through the loop with 0.5 mL of fraction volume.

  10. Collect the different factions and analyze through SDS PAGE. Store the protein at -80°C in aliquots for further use. Mix 30 µL of sample with 5× SDS gel loading dye (6 µL). Heat the sample for 10 min at 100°C before loading.

Recipes

  1. Lysis buffer (1 L)

    50 mM NaH2PO4 (6.90 g of NaH2PO4·H2O; MW 137.99 g mol-1)

    300 mM NaCl (17.54 g of NaCl; MW 58.44 g mol-1)

    10 mM Imidazole (0.68 g of Imidazole; MW 68.08 g mol-1)

    Adjust pH to 8.0 using NaOH.

  2. Wash Buffer (1 L)

    50 mM NaH2PO4 (6.90 g of NaH2PO4·H2O; MW 137.99 g mol-1)

    300 mM NaCl (17.54 g of NaCl; MW 58.44 g mol-1)

    30 mM Imidazole (2.04 g of Imidazole; MW 68.08 g mol-1)

    Adjust pH to 8.0 using NaOH.

  3. Elution Buffer (1 L)

    50 mM NaH2PO4 (6.90 g of NaH2PO4·H2O; MW 137.99 g mol -1)

    300 mM NaCl (17.54 g of NaCl; MW 58.44 g mol-1)

    300 mM Imidazole (20.4 g of Imidazole; MW 68.08 g mol -1)

  4. Buffer for Agarose Gel Electrophoresis

    TBE (Tris Boric acid EDTA) Buffer, pH = 8.0

    For 10× TBE Buffer, dissolve 108 g Tris base, 55 g Boric acid, and 7.4 g of EDTA in 750 mL of water. Adjust the pH of the solution to 8.0 and make the final volume up to one liter. 1× TBE buffer is used as the working solution.

  5. Wash Buffer

    PBS-Tween-20, pH = 7.4

    200 mL of 10× PBS

    1 mL of Tween 20

    Adjust volume to 2 L with MQ water

  6. Coating Buffer

    Carbonate Buffer, pH = 9.6

    1.59 g Na2CO3

    2.93 g NaHCO3

    0.2 g NaN3

    Adjust volume to 1 L with MQ water

  7. Counting of Expi293FTM Human Cells.

    1. Aspirate 0.1 mL of cell suspension from the single cell suspension and stain with 0.1 mL of trypan blue (0.4%).

    2. Count both Dead and live cells for percentage viability calculations.

    3. Calculations:

      Cell counting = No. of viable cells × 2 × 104 Cells per mL

  8. 10× stock of gel loading dye (10 mL):

    1. Weigh 25 mg of bromophenol blue and dissolve in 7 mL of ddH2O in a 30 mL screwcap tube.

    2. Add 2.5 g of Ficoll and dissolve it (keep in shaker overnight to allow it to dissolve completely).

    3. Measure the volume using a pipette and make up to 10 mL using sterile ddH2O. Label and store at 4°C.

    4. The final concentration would be 0.25%.

    5. Bromophenol blue and 25% Ficoll.

  9. Antibiotic concentration

    1. Stock concentration= 100 mg/mL

    2. Working concentration= 100 μg/mL

    3. Therefore, for 2 mL

    4. N1V1=N2V2

    5. X*100000 = 200*100

    6. X = 200 μL

  10. Phosphate buffered saline [pH 7.2]

    1. NaCl = 8.0 g

    2. KCl = 0.2 g

    3. Na2HPO4 = 1.15 g

    4. KH2PO4 = 0.2 g

    All the components were dissolved in 700 mL of distilled water, and the pH was checked to be at 7.2 and made up to 1,000 mL.

  11. Acrylamide:Bis (100 mL, 30% stock)

    1. Acrylamide = 29.2 g

    2. Bis acrylamide = 0.8 g

    3. ddH2O = 7 mL

      Acrylamide and Bis acrylamide were weighed and dissolved in 70 mL of water, and the volume was made up to 100 mL. The solution was filtered through Whatman No.2 paper and stored at 4°C in an amber bottle.

  12. Upper Gel Buffer [pH 6.8] 100 mL

    This is nothing but the stacking gel buffer, i.e., 0.5 Molar Tris-HCl (4×). 6.6 g of Tris base was dissolved in 70 mL of ddH2O, and pH was checked and adjusted to 6.8 using 5 N HCl. The volume was made up to 100 mL and stored at 4°C.

  13. Lower Gel Buffer [pH 8.8] 200 mL

    Separating gel buffer (1.5 M Tris-HCl (4×))

    36.3 g of Tris was dissolved in 125 mL of ddH2O. pH was adjusted to 8.8 using 5 N HCl. Volume was made up to 200 mL and autoclaved. The solution was stored at 4°C.

  14. Tank Buffer 1× (pH 8.3) 2 L

    Tris base = 6.0 g Final conc. 0.025 M

    Glycine = 28.8 g Final conc. 0.192 M

    SDS = 2 g Final conc. 0.1 M

    ddH2O = 1,750 mL

    pH was checked to be approximately 8.3 and volume was made up to 2 L.

  15. Destaining solution I

    Methanol = 250 mL (50%)

    Acetic acid = 35 mL (7%)

    ddH2O= 215 mL

  16. 2× Sample buffer (5 mL)

    1. Tris-HCl (pH 6.8) = 1.25 mL (0.12 M)

    2. SDS = 0.2 g (4%)

    3. BME = 500 µL (10%)

    4. Glycerol = 1 mL (20%)

    5. Bromophenol blue (0.15%) = 500 µL (0.015%)

    6. ddH2O = 1.75 mL

    7. Total = 5 mL

    This was dissolved into aliquots and frozen in -20°C [0.15% Bromophenol Blue is prepared by dissolving 15 mg in 0.2 mL methanol and then adding 9.8 mL of water].

  17. Western Blotting solution Preparation

    Transfer Buffer

    Tris = 0.6 g (0.025 M)

    Glycine = 2.88 g (0.192 M)

    Methanol = 40 mL (20%)

    SDS = 60 mg (0.03%)

    Volume was made upto 160 mL and autoclaved. To this, 40 mL of methanol was added and stored at 4°C.


    TBS (Tris Buffer Saline)
    (400 mL)

    Tris = 4.84 g (0.1 M)

    NaCl = 3.6 g (0.9%)

    Components were dissolved, pH was adjusted to 7.5 with HCl, and volume was made up to 400 mL.


    TTBS
    (250 mL)

    TBS = 250 mL

    Tween 20 = 250 µL


    Blocking solution
    (30 mL)

    TTBS = 12 mL

    5% Gelatin = 18 mL (3%)


    Composition of Reagents:

    Buffer P1 [Resuspension buffer]

    50 mM Tris-HCl (pH 8)

    10 mM EDTA

    100 µL/mL RNase A


    Buffer P2 [Lysis buffer]

    200 mM NaOH

    1% SDS (w/v)


    Buffer P3 [Neutralization buffer]

    3 mM potassium acetate (pH 5.5)


    Buffer QBT [Equilibrium buffer]

    750 mM NaCl

    50 mM Mops (pH 7)

    15% isopropanol (v/v)

    0.15% Triton X-100 (v/v)


    Buffer QC [ Wash buffer]

    1 mM NaCl

    50 mM Mops (pH 7)

    15% isopropanol (v/v)

Acknowledgments

We thank the Medical Research Council of the United Kingdom for allowing us to use the Tomlinson libraries. We thank Prof. S Sinha, department of Biochemistry, AIIMS, New Delhi for his critical inputs during screening of the libraries. information—This work was supported by the Department of Biotechnology and by a Translational Health Science & Technology Institute core grant.

Competing interests

There are no conflicts of interest or competing interests.

References

  1. Barderas, R., Shochat, S., Martinez-Torrecuadrada, J., Altschuh, D., Meloen, R. and Ignacio Casal, J. (2006). A fast mutagenesis procedure to recover soluble and functional scFvs containing amber stop codons from synthetic and semisynthetic antibody libraries. J Immunol Methods 312(1-2): 182-189.
  2. Borghardt, J. M., Kloft, C. and Sharma, A. (2018). Inhaled Therapy in Respiratory Disease: The Complex Interplay of Pulmonary Kinetic Processes. Can Respir J 2018: 2732017.
  3. Frenzel, A., Schirrmann, T. and Hust, M. (2016). Phage display-derived human antibodies in clinical development and therapy. MAbs 8(7): 1177-1194.
  4. Kumar, R., Andrabi, R., Tiwari, A., Prakash, S. S., Wig, N., Dutta, D., Sankhyan, A., Khan, L., Sinha, S. and Luthra, K. (2012). A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C. BMC Biotechnol 12: 87.
  5. Kumar, R., Kumari, R., Khan, L., Sankhyan, A., Parray, H. A., Tiwari, A., Wig, N., Sinha, S. and Luthra, K. (2019a). Isolation and Characterization of Cross-Neutralizing Human Anti-V3 Single-Chain Variable Fragments (scFvs) Against HIV-1 from an Antigen Preselected Phage Library. Appl Biochem Biotechnol 187(3): 1011-1027.
  6. Kumar, R., Parray, H., Narayan, N., Garg, S., Rizvi, Z. A., Shrivastava, T., Kushwaha, S., Singh, J., Murugavelu, P., Anantharaj, A., et al. (2022). A broadly neutralising monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV2 variant of concerns. Research Square. DOI: 10.21203/rs.3.rs-1431974/v1.
  7. Kumar, R., Parray, H. A., Shrivastava, T., Sinha, S. and Luthra, K. (2019b). Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol 135: 907-918.
  8. Marcus, W. D., Lindsay, S. M. and Sierks, M. R. (2006). Identification and repair of positive binding antibodies containing randomly generated amber codons from synthetic phage display libraries. Biotechnol Prog 22(3): 919-922.
  9. Parray, H. A., Shukla, S., Samal, S., Shrivastava, T., Ahmed, S., Sharma, C. and Kumar, R. (2020). Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives. Int Immunopharmacol 85: 106639.
  10. Perween, R., Ahmed, S., Shrivastava, T., Parray, H. A., Singh, B., Pindari, K. S., Sharma, C., Shukla, S., Sinha, S., Panchal, A., K. et al. (2021a). A rapid novel strategy for screening of antibody phage libraries for production, purification, and functional characterization of amber stop codons containing single-chain antibody fragments. Biotechnol Prog 37(3): e3136.
  11. Reader, R. H., Workman, R. G., Maddison, B. C. and Gough, K. C. (2019). Advances in the Production and Batch Reformatting of Phage Antibody Libraries. Mol Biotechnol 61(11): 801-815.

简介

[摘要]噬菌体展示是一种 用于选择针对所需目标的特异性抗体的成熟且广泛使用的技术。然而,需要付出巨大的努力才能从大型多样的组合文库中识别和筛选所需的阳性克隆。另一方面,从合成和半合成文库中选择阳性结合克隆对具有随机产生的琥珀终止密码子的克隆具有固有的偏见,因此更难以识别理想的结合抗体。为了克服使用琥珀密码子筛选所需克隆的问题,我们提出了一种分步方法来进行有效的噬菌体文库筛选以分离有用的抗体。该程序需要创建一个简单的新载体系统,用于在其单链抗体片段 ( scFv ) 基因序列中具有一个或多个琥珀终止密码子的噬菌体 ELISA 阳性结合克隆的可溶性生产,这在标准筛选中是困难的。

图形概要:


[背景]基于单克隆抗体的生物分子通常用于疾病检测和预防(Borghardt et al ., 2018; Parray et al ., 2020; Kumar et al ., 2022) 。单链可变片段 ( scFv ) 抗体是最常用的生物分子之一,因为它是最小的抗体单元,具有低免疫原性和低成本生产特性(Kumar等人,2019b;Parray等人,2020) . scFv是最常用的组合治疗实体,无论是单独使用还是与其他药物联合使用( Frenzel等人,2016;Kumar等人,2019b) 。 scFv形式的抗体具有可变的重链 (VH) 和轻链 (VL) 部分,它们通过可在大肠杆菌中有效产生的有效接头连接(Kumar等人,2012;Kumar等人, 2019a) 。噬菌体展示技术是所有体外展示方法中最流行和最成功的产生scFv抗体片段的方法。用于筛选的文库的大小和功能多样性提高了从噬菌体展示抗体文库中分离scFv分子的效率。来自噬菌体文库的 scFv 克隆的可溶性表达最常见的问题是 scFv 基因中琥珀密码子的频率较高,导致scFv克隆在非抑制大肠杆菌菌株中过早表达。这在合成和半合成文库的情况下更为常见,因为这些文库是在少数残基处随机构建的——特别是在 NNN、NNK、NWG、NWC 和 NSG 密码子处——这增加了琥珀密码子的偏向包含(Marcus et al ., 2006) . 由于抗体基因序列中经常存在琥珀密码子,功能性可溶性scFv分子的分离是筛选合成和半合成文库过程中遇到的最普遍的问题,导致scFv克隆在非抑制大肠杆菌中的过早表达。大肠杆菌菌株(Barderas等人,2006;Perween等人,2021a) 。然而,包含琥珀终止密码子不会影响大肠杆菌抑制菌株中噬菌体表面上scFv的展示,但会降低功能性可溶性scFv蛋白表达克隆总数的总产量。
指导重新合成单个scFv基因或使用 Kunkel 诱变是解决此问题的两种流行方法。考虑到目的是筛选大部分克隆时,这两个过程都变得昂贵且耗时,尤其是在病毒靶标中,其中大量筛选对于生成少量中和克隆至关重要(Reader等人,2019 )。
在这个生物协议中,我们描述了一种用于快速筛选含有琥珀密码子的scFv并将它们转化为可应用于多个噬菌体抗体库的可溶性scFv的新策略。我们讨论了一种快速可靠的筛选策略,该策略可用于筛选编码系列中具有琥珀终止密码子 (TAG) 的大量噬菌体抗体库。

关键字:噬菌体展示, 琥珀密码子, scFv, 高通量筛选, 新型矢量系统



材料和试剂


所有化学品均为分析试剂分子生物学/组织培养级。

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磷酸氢二钠生物试剂NIST2186IISigma-Aldrich
停止解决方案N600赛默飞世尔科技
硫酸链霉素CMS220海美达
蔗糖MB025海美达
TG1 电感受态细胞23227卢西根
TMB (四甲基联苯胺)底物溶液N301赛默飞世尔科技
三碱基TC072海美达
Tris 缓冲盐水R017R.0000赛默飞世尔
Tris 游离碱MB029-500G海美达
Tris-HClMB030海美达
海卫 X100MB031海美达
台盼蓝T8154Sigma-Aldrich
胰蛋白酶TC598海美达
吐温 20MB067海美达
•山羊亲和纯化的人 IgG Fc 抗体、碱性磷酸酶缀合的山羊亲和纯化的 IgG Fc 抗体和纯化的人 IgG 全分子购自Cappel , MP Bio, USA。
•Zolla博士赠送了一种针对细小病毒 B19 的人mAb 1418 帕兹纳, NYU SoM , 美国


塑料制品
使用的所有塑料器皿都是一次性的(这项工作中不使用玻璃器皿)。
姓名目录号公司名称
用于 ELISA 的 96 孔平底免疫板CLS3370康宁
用于 ELISA 的 96 孔圆底免疫板CLS3367康宁
96孔组织培养板CLS 3628康宁
5 mL、10 mL 和 20 mL 一次性移液器CLS4487康宁
微量离心管 1.5 mLCLS3620康宁
0.2 mL PCR 管PCR-02-A氧气
培养皿460062塔森
移液器吸头 0.5–10 μLAXYT300RS康宁
移液器吸头 1–200 μLCLS4860康宁
T-25 cm 3组织培养瓶C6231康宁
T-75 cm 3组织培养瓶C7231康宁


缓冲区
1.10 ×凝胶上样染料库存(见配方)
2.2 ×样品缓冲液(参见配方)
3.丙烯酰胺:Bis (100 mL)(参见食谱)
4.抗生素浓度(见食谱)
5.琼脂糖凝胶电泳缓冲液(见配方)
6.包衣缓冲液(见配方)
7.试剂组成(见配方)
8.Expi293F TM人类细胞计数(见食谱)
9.脱色溶液 I(见配方)
10.洗脱缓冲液 (1 L)(参见配方)
11.下凝胶缓冲液 [pH 8.8] 200 mL(参见配方)
12.裂解缓冲液(1 L)(见配方)
13.磷酸盐缓冲盐水(见配方)
14.scFv的纯化(见配方)
15.Tank Buffer 1 × 2L (pH 8.3)(见配方)
16.上凝胶缓冲液 [pH 6.8] 100 毫升 (见食谱)
17.洗涤缓冲液(1 L)(见配方)
18.洗涤缓冲液(见配方)
19.Western Blotting 溶液(见配方)


程序


A.辅助噬菌体生产
辅助噬菌体对于将噬菌粒颗粒转移到大肠杆菌中是必需的。噬菌粒颗粒包含 ( i ) 抗生素制造商,(ii) 抗体-G3P 融合蛋白,和 (iii) 噬菌体复制起点。
噬菌粒文库与抗体-G3P 融合蛋白和辅助噬菌体基因一起扩增,这些基因是感染、复制、组装和出芽所必需的。


1.取三个 Corning ® 50 mL Falcon 离心管 (FCT) 并将它们标记为 A、B 和 C。
2.在每个 FCT 管中添加 5 mL 的 2 × YT 介质。
3.以 B 和 C 作为阴性对照,其中一个加入氨苄青霉素,另一个加入卡那霉素。
4.添加 20 μL的 TG1大肠杆菌细胞。
5.将 FCT 在 37°C 下摇动孵育过夜生长。
6.5 mL 新鲜的 2 × YT 培养基中继代培养 20 μL先前接种的 TG1 细胞在FCT A(步骤A1)中;孵育 2 – 4 小时 在 37°C 下摇动。
7.在培养的 TG1 细胞中加入 40 μL的辅助噬菌体。
8.在 37°C 下生长 30 分钟,不要摇晃。
9.在 37°C 下振荡培养 30 分钟。
10.将此培养物添加到 200 mL 的新鲜 2 × YT 培养基中,其中含有 50 μg / mL 工作浓度的卡那霉素。
11.允许在 30°C 下摇晃过夜生长。
12.从培养箱中取出烧瓶,将培养物收集在高压灭菌的铯瓶中。
13.14,260 × g离心机 在 4°C 下 30 分钟。
14.将上清液倒入另一个新鲜的铯瓶中,以 14,260 × g离心 和 4°C 30 分钟。
15.在不干扰颗粒的情况下,将上清液收集在玻璃瓶中,加入 PEG/NaCl [20% ( wt /vol) 聚乙二醇 6000, 2.5 M NaCl],保持上清液和 PEG/NaCl 的比例为 50:15。
16.在 4°C 的冷藏室中存放 4 – 5 小时,或过夜以获得更好的效果。
17.以 14,260 × g旋转培养物 和 4°C 1 小时,让细胞沉淀下来。
18.在不干扰颗粒的情况下,丢弃上清液,并用 1 × PBS 重新悬浮颗粒,将其保存在 1.5 mL 管中。
19.以 16,200 × g离心微管 5分钟。如果形成沉淀,将上清液转移到新试管中并储存在 4°C(图 1)。




 
图1。 辅助噬菌体制备步骤的示意图。


B.成长汤姆林森 I + J 并制作二级股票
1.取 100 mL 的 2 × YT 培养基,其中含有氨苄青霉素和 1%(体积/体积)葡萄糖。在此介质中,添加 500 μL的 Tomlinson I + J 噬菌体库库存。
2.孵育 1 – 2 小时,直到 600 nm 处的 OD 约为 0.4。
3.将 100 mL 的培养基分成两部分。首先,使用 50 mL 培养文库;然后,使用剩余的媒体制作图书馆的二级库存。


培养图书馆(噬菌体库存):
4.取 100 mL 生长培养基中的 50 mL,加入 200 μL辅助噬菌体。
5.在 37°C 下孵育30 分钟,不要摇晃。
6.1,200 × g离心 10分钟。
7.小心丢弃上清液,不要干扰沉淀。
8.将颗粒溶解在 100 mL 的 2 × YT 介质中,该介质含有 100 μg / mL 氨苄青霉素、50 μg / mL 卡那霉素和 0.1%(重量/体积)葡萄糖。
9.将重悬的沉淀在 30°C 下摇动孵育过夜。
10.过夜孵育后,将培养物转移到离心瓶(铯瓶)中并以 14,260 × g离心 和 4°C 30 分钟。
11.将上清液转移到另一个铯瓶中,再次以 14,260 × g离心 和 4°C 30 分钟。
12.小心地将上清液转移到另一个高压灭菌的玻璃瓶中并丢弃沉淀。将 PEG/NaCl(20% 聚乙二醇 6000、2.5 M NaCl)添加到收集的上清液中(15 mL 的 PEG/NaCl 到 50 mL 上清液)。
13.将其存放在 4°C 的冷藏室中 4 – 5 小时,或过夜以获得更好的效果。
14.以 14,260 × g旋转培养物 和 4°C 1 小时,让细胞沉淀下来。
15.在不干扰沉淀的情况下,弃去上清液并用 1 × PBS 重悬沉淀;将其保存在 1.5 mL 管中。
16.× g f或 10 分钟离心微管。如果形成沉淀,将上清液转移到新鲜的试管中,并在 4°C 下短期储存。添加 15% (vol/vol) 甘油可在 -80°C 下更长时间储存(图 2)。


制作噬菌体文库二级储备:
17.将剩余的 50 mL 培养基在 37°C 下摇动培养 2 小时。
18.在 1,500下离心培养物,让细胞沉淀下来 × 克 15分钟。
19.含有 15% (vol/vol) 甘油的 3 mL 2 × YT 介质中重新悬浮颗粒。
20.储存在 -80°C 直至进一步使用(图 2)。


 


图 2.用于筛选目的的文库扩增和文库二级库存制备所涉及步骤的示意图。


C.生物平移
在这一步中,目标蛋白被固定在微量滴定板的表面。第一步是添加获救的汤姆林森噬菌体文库。第二步涉及结合,其中展示最高亲和力抗体的scFvs的噬菌体结合抗原的表位,而具有低结合亲和力的那些通过洗涤去除。通过使用胰蛋白酶的酶消化来洗脱结合抗原的噬菌体。洗脱的噬菌体感染TG1,然后添加辅助噬菌体进行扩增。为了积累展示高亲和力抗体片段的噬菌体,这些步骤对前一轮淘选的扩增噬菌体重复3次,每次都增加洗涤循环的次数。


第1轮
1.实验前一天,在ELISA板的一排(比如C排),即A板,涂上所需的抗原(每孔100 μL ),浓度为5 μg 。用包被缓冲液包被抗原。
2.将板 A 在 4°C 下孵育过夜。
3.第二天,在 5 mL PBS 中制备 3%BSA,并在 37°C 下孵育 10 分钟。然后,涂上另一块板,即板B;涂上两排(例如 C 和 D 排)并在 37°C 下孵育板 1 小时。
4.μL的噬菌体原液中涂上一小撮脱脂牛奶(比如 C 行) 。每口井涂 100 μL 。
5.在室温下孵育板 C 30 分钟。然后,将涂层从 C 行转移到任何其他行,例如 D 行。再孵育30 分钟。
6.涂板 B 1 小时后,用高压灭菌 PBS(每孔 250 μL )清洗一次板,并将板 C 的内容转移到板 B 的 C 行上。
7.再次,在室温下孵育板 B 30 分钟,然后将 C 行的内容转移到 D 行,然后再孵育 30 分钟。
8.在清洗板 B 的同时,使用高压灭菌的 PBS(每孔 250 μL )清洗板 A,然后用 3%(重量/体积)BSA(每孔 200 μL )封闭。在室温下孵育 1 小时。
9.用高压灭菌的 PBS 清洗板 A 三次。
10.然后,将板 B 的内容转移到板 A 上;然后在室温下孵育 1 小时。
11.孵育后,用 PBST 清洗板 A 10 次。制作 1 mL 的 PBS,其中含有 50 μL的胰蛋白酶,并在每口井中加入 95 μL这种溶液。
12.将板在 37°C 下保持 10 分钟。
13.胰蛋白酶化的内容收集到一个等分试样中。这将是第一轮生物淘选的输出。
14.使用生物平移 1 的输出来计算换能单元 (T U) (图 3)。


 
图 3.生物淘选过程中涉及的步骤的示意图。


准备下一轮生物淘选
15.将 500 μL的生物平移输出添加到 5 mL 的 TG1 细胞生长培养基中。
16.在 37°C 下振荡保持 30 分钟。
17.以700 × g离心10 分钟。
18.使用 1 mL 上清液溶解离心过程中形成的沉淀,并将剩余的上清液倒出。
19.将其涂抹在含有 2 × YT 琼脂和氨苄青霉素的生物测定皿上。
20.让细菌在 37°C 下过夜生长。
21.第二天,将 3 – 5 mL 的含有 15% 甘油的 2 × YT 培养基添加到生物测定皿中,并刮掉所有过夜生长的菌落。将它们收集在新管中。
22.使用 100 μL的刮去菌落并储存其余部分。
23.含有氨苄青霉素 (100 µg/mL) 和 1% (vol/vol) 葡萄糖的 50 mL 2 × YT 介质中加入 100 μL的刮除菌落。
24.让它在 37°C 下振荡生长 2 小时。
25.取上述培养物 10 mL,加入 40 μL辅助噬菌体。在 37°C 下孵育 30 分钟,不要摇晃。
26.以 700 × g离心培养15 分钟,弃去上清液,不要干扰沉淀。
27.将颗粒溶解在 50 mL 的 2 × YT 介质中,其中含有 100 μg / mL 氨苄青霉素、50 μg / mL 卡那霉素和 0.1% 葡萄糖。在 30°C 下孵育过夜 颤抖着。
28.将过夜培养的培养物在 14260 × g和 4°C 下离心 30 分钟,将上清液收集在新鲜的铯瓶中,并在 14260 × g和 4°C 下再次离心 30 分钟。
29.小心地将上清液转移到高压灭菌的玻璃瓶中并丢弃沉淀。将 PEG/NaCl(20% 聚乙二醇 6000、2.5 M NaCl)添加到收集的上清液中(15 mL 的 PEG/NaCl 到 50 mL 的上清液)。
30.将其存放在 4°C 的冷藏室中 4-5 小时,或过夜以获得更好的效果。
31.14260 × g旋转培养物1 小时,使细胞沉淀下来。
32.在不干扰颗粒的情况下,丢弃上清液并用 1 × PBS 重新悬浮颗粒,将其保存在 1.5 mL 管中。
33.× g将微管离心10 分钟。如果形成沉淀,将上清液转移到新试管中并储存在 4°C。
34.收集的噬菌体将用作下一轮生物淘选的输入,方法是将其涂在板 C 上,而不是噬菌体原液。
注意:每轮生物淘选,降低抗原包被浓度(例如,第 1 轮使用 5 μg / μL ,第 2 轮使用 3 μg / μL , 并用 1.5 μg / μL进行第 3 轮)并计算噬菌体选择期间使用的每个输入和输出的 TU。


D.噬菌体 ELISA 筛选
第 0 天:进行 ELISA 前一天
1.对于噬菌体选择过程,培养最后一轮生物淘选输出的菌落。接种在最后一轮生物淘选输出中形成的菌落;每次接种均在 5 mL含有 100 μg / mL 氨苄青霉素的 2 × YT 培养基中进行。
2.在 37°C 下孵育 30 分钟,不要摇晃。等到文化的 OD 600达到 0.4–0.6。
备份集:在此步骤中,取每个克隆的200 μL培养物,并加入 200 μL高压灭菌的 50% (vol/vol) 甘油溶液制成储备液并储存在 -80°C 以供将来实验
3.添加 20 μL的辅助噬菌体。
4.再次在 37°C 下摇动孵育 30 分钟。
5.加入 50 μg / mL 卡那霉素并在 30°C 下以 140 – 160 × g振荡孵育过夜。
6.μg / μL的所需抗原涂抹 96 孔检测板。将 BSA 涂层作为阴性对照,并将板在 4 °C 下过夜(图 4)。


 
图 4.噬菌体 ELISA 筛选过程中涉及的步骤的示意图。


酶联免疫吸附试验
7.第二天,将抗原包被板从 4°C 中取出并用 PBS 洗涤一次。
8.中 200 μL的 5% 脱脂牛奶块板。
9.在室温下孵育板 1 小时。
10.将所有菌落接种物从培养箱中取出,以 3,900 × g离心管20分钟。
11.μL )清洗 ELISA 板 3 次。
注意:如果需要,阻塞可以延长到 90 分钟,具体取决于并行步骤的时间
12.孵育 1 小时后,用 PBS 洗板 3 次。
13.然后每孔加入100 μL一抗,即从所有离心管中取50 μL上清液和50 μL脱脂牛奶(按1:1稀释)。
14.在室温下孵育 1 小时。
15.用 250 μL的 0.1% PBST 清洗盘子四次。
16.加入 100 μL的二级抗体(1:2,000)。随后在黑暗中在室温下孵育 1 小时。
17.用 250 μL的 0.1% PBST 清洗盘子六次。
18.加入 100 μL的底物 (TMB)。
19.让反应在黑暗中进行 15 – 20 分钟。
要停止反应,每孔加入 50 μL停止溶液 (2 NH 2 SO 4 ),并在多模式 ELISA 阅读器上以 450 nm 读取板。


E.质粒 DNA 的分离
1.在噬菌体 ELISA 中识别阳性结合克隆(至少是阴性对照的四倍)。
2.取两个 FCT 并将它们标记为 A 和 B;向每个 FCT添加 5 mL 的 2 × YT 介质。
3.取 B 并添加氨苄青霉素 (100 µg/mL) 和 C 作为阴性对照,在其中添加卡那霉素 (50 µg/mL)。
4.在 FCT A 和 B 中,从保存在 -80°C 的甘油原液中接种。
5.将 FCT 在 37°C 下摇动孵育过夜生长。
6.第二天早上,检查管 A 和 B。管 A 培养物应该是混浊的,管 B 中应该没有生长。
7.通过在 2,820 × g下将培养物离心15 分钟来降速。
8.对于质粒分离,请按照制造商的说明使用 Qiagen Miniprep 试剂盒。
9.使用 nanodrop 分光光度计检查分离质粒 DNA 的质量和浓度。分离 DNA 的 260/280 比率应为 1.8。
10.准备 0.8% 琼脂糖凝胶并检查凝胶上分离质粒 DNA 的质量。
11.制作 10 μL的质粒 DNA 等分试样,并使用 LMB3 和 PHEN 测序引物对scFv插入序列进行测序。


可溶性ELISA
12.如噬菌体 ELISA 部分所述执行 ELISA。
13.加入 100 μL纯化的scFv ,在室温下孵育 1 小时。
14.用 0.1% PBST洗板3 次。
15.在 2% MPBS 中使用 1:1,000 稀释的初级抗体(抗 His 标签)并在室温下孵育。
16.0.1% PBST洗涤 3 次。
17.如上所述,在 2% MPBS 中使用 1:2,000 稀释的抗兔 HRP 偶联二抗并在室温下孵育,然后洗涤。
18.添加 100 μL 的 TMB 底物,让颜色显色。一旦颜色出现,加入 8 NH 2 SO 4以停止反应。
19.在 ELISA 阅读器中读取 450 nm 处的板。


F.用于转换单位 (TU) 计算的稀释和电镀


 
图 5.显示辅助噬菌体/文库 TU 计算的稀释制备策略的代表性图像。


1.每个稀释液的铺板在含有氨苄青霉素的不同板上进行。
2.将噬菌体和细菌的混合物倒在盘子上,并使用吊具缓慢扩散。用它包含的稀释度标记每个板。
3.将板在 37°C 下孵育过夜生长。第二天,计算 TU 计算的菌落(图 5) 。


G.计算换能器
TU = (菌落数 × 1000) /(10 × 稀释度)。对于 10 -8稀释板,我们得到了 9 个菌落;
那么TU计算为: (9 × 1000) /( 10 × 10 -8 ) = 9 × 10 10


H.感受态细胞的制备
1.划线大肠杆菌TG1 细胞,让细胞在 37°C 下过夜生长。
2.在 5 mL LB 培养基中接种单个菌落,并在 37°C 下生长过夜。
3.通过接种 1 mL 过夜培养的大肠杆菌,在 100 mL LB 中进行继代培养,并在 37°C 的摇床上生长,直到 600 nm 处的 OD 达到约 0.6。
4.立即在冰上冷却培养物,并通过在 4,200 × g和 4°C 下离心 5 分钟来收获细胞。
5.小心取出上清液;将离心管倒置在纸巾上,去除任何上清液痕迹。
6.将细菌颗粒重新悬浮在 10 mL 的冰冷 0.1 M CaCl 2 (高压灭菌)中,并在冰上孵育 30 分钟。
7.如上所述通过离心回收细胞,重悬于 5 mL 的 0.1 M CaCl 2中,并在微量离心管中分装 200 μL细胞。


I.矢量设计
图 6 显示了矢量设计的代表性策略。
一组设计的 PCR 引物在scFv-pIII连接处将 TAG 琥珀密码子替换为 TAA,用于载体构建。
正向引物:5'CACATCATCATCACCATCACGGGTAATAAGAACAAAAACTCATCTC3'
反向引物:5'GAGATGAGTTTTTGTTCTTATTACCCGTGATGGTGATGATGATGTG3'。
PCR反应设置:
脚步初始变性骑行 16 ×扩大抓住
温度95°C95°C52°C72°C72°C10°C
时间2 分钟30 秒50 秒4 分钟5分钟∞


PCR产物被Dpn 1酶消化
PCR产物20 µL
Dpn 1酶1 µL
剪切智能缓冲区2.5 µL
将反应混合物在 37°C 下孵育 2 小时。轻敲中间的管子。


1.将两瓶感受态细胞从 -80°C 冰箱中置于冰上解冻 5-10 分钟。不要在这一步点击。
2.将 5 μL 的Dpn 1 消化产物添加到一个管中,并将另一管保持为空白或阴性对照。在冰上孵育细胞 30 分钟。
3.30 分钟后,将两根管子放入浮子中热休克 60 秒(在此步骤中,将水浴设置为 42°C)。
4.立即将试管置于冰中 5 分钟;在此步骤中,将介质预热至 37°C。
5.在管中的细胞中加入 900 μL 的预热 2 × YT 培养基。
6.以 220 rpm 的速度在摇床中孵育管子 60 分钟以生长细胞。
7.从摇床中取出管子,吸出 100 μL 的细胞悬浮液,并在 LB-Agar-氨苄青霉素板上进行平板/涂抹。将板在 37°C 培养箱中孵育 16 小时或过夜。
8.第二天早上,取出平板,计数菌落,并在 4°C 下储存直至进一步使用。
9.将 5 mL 的 2 × YT 添加到五管中,并辅以标准浓度的氨苄青霉素。挑取单个菌落对平板上获得的菌落进行接种培养;在 37°C 下以 220 rpm 振荡孵育。还孵育一管培养基作为对照。
10.第二天早上,按照制造商的说明,使用 Qiagen 质粒分离试剂盒从所有四个管中分离质粒 DNA。
11.通过观察 260/280 DNA 比率,使用分光光度计检查分离质粒的质量。
12.分装 DNA 并将这些样本发送到使用载体特异性引物进行测序。
13.分析点突变的 DNA 序列数据并标记阳性克隆以供进一步使用。丢弃阴性克隆。


设计载体的消化
限制性消化的反应混合物:
零件体积( μL )
质粒 DNA (250 ng · μL -1 )10微升
10 × CutSmart 缓冲器 (NEB)5微升
Nco I -HF (NEB)2微升
不是I -HF (NEB)2微升
无核酸酶水31微升
将反应在 37°C 下孵育 2 小时。


1.2 小时后,加入 10 μL的 5 ×凝胶上样染料,并在 0.8% 琼脂糖凝胶上以 100 V 运行样品约 60 分钟。
2.使用锋利的手术刀片从琼脂糖凝胶中切割和切除消化的载体 DNA。将切除的片段放入 1.5 mL Eppendorf 管中。根据制造商的协议,使用 Qiagen 凝胶提取试剂盒从切除的 DNA 中纯化消化的载体。
3.通过计算 260/280 比率,使用分光光度计评估纯化的消化载体 DNA 的质量。这种纯化的载体用于未来的克隆反应。


scFv克隆的消化
进一步使用在可溶性ELISA中未显示结合的噬菌体ELISA结合阳性克隆从单菌落中分离质粒DNA 。
消化反应设置如下:
零件体积( μL ) _
质粒 DNA (250 ng · μL -1 )10微升
10 × CutSmart 缓冲器 (NEB)5微升
Nco I -HF (NEB)2微升
不是I -HF (NEB)2微升
无核酸酶水31微升


1.2 小时后,加入 10 μL的 5 ×凝胶上样染料,并在 1% 琼脂糖凝胶上以 100 V 运行样品约 40-60 分钟。
2.在琼脂糖凝胶中应观察到两条条带,一条条带大小约为 4 kb,对应于载体 DNA,另一条条带大小为 800 bp,对应于scFv DNA。
3.切除含有相关 DNA 片段的琼脂糖凝胶切片( scFv插入 800 bp)并去除多余的琼脂糖以最小化凝胶切片。
4.根据制造商的说明,将凝胶切片转移到微量离心管中并使用 Qiagen 凝胶提取试剂盒进行纯化。
5.使用这种纯化的scFv DNA 克隆到新设计的载体中。


scFv DNA克隆到新设计的载体中
零件体积( μL ) _
scFv DNA插入10微升
设计的矢量5微升
10 ×连接酶缓冲液2微升
连接酶2微升
无核酸酶水31微升


1.将连接反应混合物在室温下孵育 4-6 小时。或者,这可以在室温下保持过夜。
2.后,将 TG1 感受态细胞在冰上解冻 5 分钟。
3.将 5 μL的结扎混合物添加到一个管中,并保持第二个管没有插入和/或结扎混合物作为阴性对照。
4.热休克细胞 60 秒,并立即在冰上保持 5 分钟。
5.加入 900 μL的 2 × YT 培养基,在 37°C 的摇床培养箱中孵育试管 1 小时。
6.× g离心管5 分钟。倒出上清液并将颗粒重新悬浮在扩孔剩余介质中。将其置于预热的 2 × YT-Agar 平板上,添加氨苄青霉素,或者您可以选择使用带有氨苄青霉素的 LB-Agar 平板。
7.将板在 37°C 培养箱中孵育过夜或 12-16 小时。
8.第二天早上,计数反应板上的菌落。控制板中不应有菌落;否则,用所有新的和新制备的试剂重复实验。
9.在两个 50 mL FCT 中接种含有标准浓度氨苄青霉素的5 mL 2 × YT 中的单个菌落。将一根管标记为反应管,另一管标记为空白管,并在 37°C 的摇床培养箱(200 至 220 rpm)中孵育两管过夜。
10.将小接种物(约 4 mL)从过夜原代培养物转移到 2 L 烧瓶(400 mL 培养基)2 × TY 中,其中含有 100 μg /mL 氨苄青霉素和 0.1% 葡萄糖。在 37°C 下逐渐摇动 (250 rpm),直到 OD 600约为 0.9(约 3 至 3.5 小时)。
11.一旦培养物的 OD 达到 0.9,加入异丙基 β-D-硫代半乳糖苷,最终浓度为 1 mM IPTG。继续在 30°C 下摇动 (250 rpm) 过夜。
12.× g下离心15 分钟来收获细胞。如果需要,将细胞沉淀储存在 -20°C 或立即处理。


 
图 6. 修改后的矢量设计策略的示意图。
用琥珀终止密码子对scFv基因进行可溶性表达的改进方法。原始载体中scFv-pIII基因之间的琥珀终止密码子 (TAG)被更改为 TAA,从而阻止了scFv-pIII融合蛋白的产生。使用用于将scFv基因克隆到原始载体中的相同限制性位点 Nco1/Not1将scFv基因直接克隆到修饰载体中(Perween等人,2021a) 。


重悬细胞沉淀和细胞提取物制剂
a.使用 NaOH 将 pH 值调节至 8.0 。
b.将颗粒重新悬浮在 30 mM Tris-HCl、20%(重量/体积)蔗糖、pH 8.0、湿重 80 mL/g 中。在冰上孵育并滴加 500 mM EDTA 至终浓度为 1 mM;然后将细胞在冰上孵育 20 分钟,同时轻轻搅拌。
c.× g和 4°C下离心 20 分钟来澄清细胞悬液。
d.收集上清液并将细胞重新悬浮在相同体积的冰冷 5 mM MgSO 4中,并在冰上孵育 20 分钟,同时轻轻搅拌。
e.× g和 4°C 下将细胞离心20 分钟。收集上清液(上清液是含有周质蛋白的渗透性休克液)并针对裂解缓冲液进行广泛透析。
f.在继续纯化之前,通过0.2 μm过滤器过滤透析的上清液。在使用 Ni++ 离子之前,用裂解缓冲液(50 mM NaH 2 PO 4、300 mM NaCl、10 mM 咪唑和 pH = 8.0)平衡树脂。


scFv的纯化
a.对于纯化,使用 Ni-NTA 珠子;在纯化柱中加入 5 mL 的 50% Ni-NTA-琼脂糖树脂浆液,让珠子沉淀下来。
b.MilliQ (MQ) 水清洗珠子;添加 10 列体积 (CV) 的 MQ。
c.使用 10 CV 的结合缓冲液(20 mM Tris;pH = 7.2、500 mM NaCl 和 10 mM 咪唑)平衡柱。因此,与 Ni++ 离子树脂相同的 pH 值和缓冲液成分可确保样品正确结合
d.将过滤后的上清液加入柱中,使其通过重力。
e.上清液通过后,清洗柱子以去除任何杂质。添加 20 – 30 CV 的洗涤缓冲液(20 mM Tris;pH = 7.2、500 mM NaCl 和 25 mM 咪唑)。
f.从珠子中洗脱结合的蛋白质,并使用 5 – 6 CV 的洗脱缓冲液(20 mM Tris pH = 7.2、500 mM NaCl 和 500 mM 咪唑)。在 15% 三甘氨酸 SDS-PAGE 上分析纯度方面的蛋白质质量。
g.使用激活的透析管透析蛋白质,将管的两端紧紧夹住,然后将其放入装有冷 PBS 的烧杯中。在磁力搅拌器的帮助下,让它在 4°C 的冷室中旋转过夜。
h.在 AKTA FPLC 系统中安装 Superdex75 增加柱。用 PBS 彻底清洗泵。
i.使用 2 CV 的 PBS 平衡色谱柱;通过循环加载大约 500 μL的纯化浓缩蛋白,体积为 0.5 mL。
j.收集不同的派系并通过SDS PAGE进行分析。将蛋白质分装在 -80°C 下储存以备进一步使用。将 30 μL 的样品与 5 × SDS 凝胶加载染料(6 μL)混合。加载前在 100 °C 下加热样品 10 分钟。


食谱


1.裂解缓冲液 (1 L)
50 mM NaH 2 PO 4 (6.90 g NaH 2 PO 4 ·H 2 O;MW 137.99 g mol -1 )
300 mM NaCl(17.54 g NaCl;MW 58.44 g mol -1 )
10 mM 咪唑(0.68 g 咪唑;MW 68.08 g mol -1 )
使用 NaOH 将 pH 值调节至 8.0。
2.洗涤缓冲液 (1 L)
50 mM NaH 2 PO 4 (6.90 g NaH 2 PO 4 ·H 2 O;MW 137.99 g mol -1 )
300 mM NaCl(17.54 g NaCl;MW 58.44 g mol -1 )
30 mM 咪唑(2.04 g 咪唑;MW 68.08 g mol -1 )
使用 NaOH 将 pH 值调节至 8.0。
3.洗脱缓冲液 (1 L)
50 mM NaH 2 PO 4 (6.90 g NaH 2 PO 4 ·H 2 O;MW 137.99 g mol -1 )
300 mM NaCl(17.54 g NaCl;MW 58.44 g mol -1 )
300 mM 咪唑(20.4 g 咪唑;MW 68.08 g mol -1 )
4.琼脂糖凝胶电泳缓冲液
TBE(Tris 硼酸 EDTA)缓冲液,pH = 8.0
对于 10 × TBE 缓冲液,将 108 g Tris碱、55 g 硼酸和 7.4 g EDTA 溶解在 750 mL 水中。将溶液的 pH 值调节至 8.0,使最终体积达到 1 升。 1 × TBE 缓冲液用作工作溶液。
5.洗涤缓冲液
PBS-Tween-20,pH = 7.4
200 毫升 10 × PBS
1 毫升吐温 20
用 MQ 水将体积调整到 2 L
6.涂层缓冲液
碳酸盐缓冲液,pH = 9.6
1.59 克 Na 2 CO 3
2.93 克碳酸氢钠3
0.2 克 NaN 3
用 MQ 水将体积调节至 1 L
7.Expi293F TM人类细胞的计数。
a.从单细胞悬浮液中吸出 0.1 mL 的细胞悬浮液,并用 0.1 mL 的台盼蓝(0.4%)染色。
b.计算死细胞和活细胞以计算百分比活力。
c.计算:
细胞计数 = 活细胞数 × 2 × 10 4细胞/mL
8.10 ×凝胶上样染料 (10 mL):
a.在 30 mL 螺旋盖管中称量 25 mg 溴酚蓝并溶解在 7 mL 的 ddH 2 O 中。
b.加入 2.5 g Ficoll并溶解(在摇床中放置过夜,使其完全溶解)。
c.2 O补足 10 mL 。标记并储存在 4 °C 。
d.最终浓度为 0.25%。
e.溴酚蓝和 25% Ficoll 。
9.抗生素浓度
a.库存浓度 = 100 毫克/毫升
b.工作浓度= 100 μ g /mL
c.因此,对于 2 mL
d.N1V1=N2V2
e.X*100000 = 200*100
f.X = 200微升 
10.磷酸盐缓冲盐水 [pH 7.2]
•氯化钠 = 8.0 克
•氯化钾= 0.2 克
•Na 2 HPO 4 = 1.15 克
•KH 2 PO 4 = 0.2 克
将所有组分溶解在 700 mL 蒸馏水中,检查 pH 值为 7.2 并定容至 1,000 mL。
11.丙烯酰胺:双(100 mL,30% 库存)
a.丙烯酰胺 = 29.2 克
b.双丙烯酰胺 = 0.8 g
c.ddH 2 O = 7 毫升
称取丙烯酰胺和双丙烯酰胺,溶于70mL水中,定容至100mL。将溶液通过 Whatman No.2 纸过滤并在 4 °C 下储存在琥珀色瓶中。
12.上凝胶缓冲液 [pH 6.8] 100 mL
这不过是浓缩胶缓冲液,即., 0.5 摩尔 Tris-HCl (4 × )。将 6.6 g Tris 碱溶解在 70 mL ddH 2 O 中,检查 pH 值并使用 5 N HCl 将其调节至 6.8。将体积定容至 100 mL 并在 4 °C 下储存。
13.下凝胶缓冲液 [pH 8.8] 200 mL
分离凝胶缓冲液 (1.5 M Tris-HCl (4 × ))
将 36.3 g Tris 溶解在 125 mL ddH 2 O 中。使用 5 N HCl 将 pH 调节至 8.8。体积定容至 200 mL 并高压灭菌。将溶液储存在 4 °C 。
14.罐缓冲液 1 × ( pH 8.3) 2 L
Tris碱 = 6.0 g最终浓度0.025 M
甘氨酸 = 28.8 g最终浓度0.192 米
SDS = 2 g最终浓度0.1M
ddH 2 O = 1,750 毫升
检查 pH 值约为 8.3,并将体积定容至 2 L。
15.脱色液 I
甲醇 = 250 毫升 (50%)
乙酸 = 35 毫升 (7%)
ddH 2 O= 215 毫升
16.2 ×样品缓冲液 (5 mL)
•Tris-HCl (Ph 6.8) = 1.25 mL (0.12 M)
•SDS = 0.2克 (4%)
•BME = 500 µL (10%)
•甘油= 1 毫升 (20%)
•溴酚蓝 (0.15%) = 500 µL (0.015%)
•ddH 2 O = 1.75毫升
•总计 = 5 毫升
将其溶解成等分试样并在 -20°C 中冷冻[通过将 15 mg 溶解在 0.2 mL 甲醇中,然后加入 9.8 mL 水来制备 0.15% 溴酚蓝]。
17.Western Blotting 溶液制备
传输缓冲器
Tris = 0.6 g (0.025 M)
甘氨酸 = 2.88 g (0.192 M)
甲醇 = 40 毫升 (20%)
SDS = 60 毫克 (0.03%)
体积增至160 mL 并高压灭菌。向其中加入 40 mL 甲醇并在 4°C 下储存。


TBS(Tris 缓冲盐水) ( 400 毫升)
Tris = 4.84 克 (0.1 M)
氯化钠 = 3.6 克 (0.9%)
将组分溶解,用 HCl 将 pH 调节至 7.5,并定容至 400 mL。


TTBS (250 毫升)
TBS = 250 毫升
吐温 20 = 250 µL


封闭溶液(30 毫升)
TTBS = 12 毫升
5% 明胶 = 18 毫升 (3%)


试剂组成:
缓冲液 P1 [重悬缓冲液]
50 mM Tris-HCl (pH 8)
10 毫米乙二胺四乙酸
100 µL/mL RNase A


缓冲液 P2 [裂解缓冲液]
200 毫米氢氧化钠
1% SDS (w/v)


缓冲液 P3 [中和缓冲液]
3 mM 醋酸钾 (pH 5.5)


缓冲液 QBT [平衡缓冲液]
750 毫米氯化钠
50 毫米拖把 (pH 7)
15% 异丙醇 (v/v)
0.15% Triton X-100 (v/v)


缓冲液质量控制 [ 洗涤缓冲液]
1 毫米氯化钠
50 毫米拖把 (pH 7)
15% 异丙醇 (v/v)


致谢


请承认支持这项工作的资金来源;如果此协议是从以前的工作中改编或修改的,也请在此处确认以前的工作。


利益争夺


不存在利益冲突或竞争利益。


参考


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引用:Singh, V., Garg, S., Raj, N., Lukose, A., Jamwal, D., Perween, R., Dhyani, S., Parray, H. A., Sharma, C. and Kumar, R. (2022). Protocol for High Throughput Screening of Antibody Phage Libraries. Bio-protocol 12(12): e4450. DOI: 10.21769/BioProtoc.4450.
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