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Summer Research Fellowship Programme of India's Science Academies

Screening of yeast surface display Nanobody Library (NbLib) for selection of PIP4K2a interacting nanobodies

Surabhi Sinha

Indian Institute of Technology Roorkee, Uttarakhand

Dr. Vasudevan Seshadri

National Center for Cell Science, Pune

Abstract

Nanobodies (Nb), or single domain antibodies provide the remarkable specificity of antibody within a single 15kDa immunoglobulin VHH domain. The ability of these nanobodies to specifically recognize unique epitopes with subnanomolar affinity has made them a useful class of biomolecules for research and other therapeutic applications. Phosphatidylinositol-5-phosphate 4-kinase type-2 alpha (PIP4K2a) is an enzyme that converts PI5P to PI-4,5-P. This protein has been recently found to be a novel RNA-binding protein by our lab. This project will help us isolate PIP4K2a-specific nanobodies for further PIP4K2a pull down assays. For selection and separation of PIP4K2a and PIP4K2a-specific yeast Nb, magnetic beads have been used as these are an ideal tool for immobilizing molecules like proteins or nucleic acids on a solid phase.

Keywords: yeast surface display, Nanobody Library, magnetic beads, pull down assays, transformation

Abbreviations

Abbreviations
 NbNanobody 
NbLib Nanobody Library 
 PI5PPhosphatidylinositol-5-phosphate 
 PI-4,5-PPhosphatidylinositol-4,5-phosphate 
 PIP4K2aPhosphatidylinositol-5-phosphate 4-kinase type-2 alpha 
 HisHistidine 
 HQ tagHistidine Glutamine tag 
 NiNickel 
 FabFragment Antigen Binding 
 VHVariable domain of heavy chain 
 VLVariable domain of light chain 
 IgGImmunoglobulin class G 
PDB Protein Data Bank 
UTR Untranslated Region 
 EDTAEthylenediaminetetraacetic acid 
 GDWGlass distilled water 
 SD MediaSynthetic defined media  
 DMSO Dimethyl sulfoxide
BSA Bovine Serum Albumin 
 HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 
LB Lysogeny Broth 
 SDS Sodium Dodecyl Sulfate
 PCRPolymerase Chain Reaction 

INTRODUCTION

Background

Antibodies have emerged as an important class of biomolecules due to their remarkable specificity and biochemical versatility. These antibodies are composed of two heavy and two light chains. The variable domain of the heavy and light chain, termed VH and VL respectively, contributes to their unique property of antigen-binding specificity. A key exception to this general architecture is found in camelids (llamas, camels, alpacas, and their relatives), which possess a parallel antibody repertoire composed solely of heavy chains.1,2 Such antibodies bind to their target antigens through a single variable domain, termed VHH, which contains the entire antigen-binding surface. Unlike the antigen binding fragments of conventional antibodies (Fabs), these isolated VHH domains (also called “nanobodies”) can be readily expressed in bacteria as the product of a single gene, and in many cases these fragments can even fold and retain antigen specificity in the reducing environment of the eukaryotic cytosol.3 This special IgG subclass is capable of binding to common antigenic determinants, peptides and small haptens. The ability of these nanobodies to specifically recognize unique epitopes with subnanomolar affinity have made them a useful class of biomolecules for research and other therapeutic applications. Here, a fully synthetic yeast display nanobody library has been used, devised using an alignment of structurally characterized nanobodies from the Protein Data Bank (PDB).

Phosphatidylinositol-5-phosphate 4-kinase type-2 alpha (PIP4K2a) is an enzyme that converts PI5P to PI-4,5-P. This protein has been recently found to be a novel RNA-binding protein by this institute. It has been found to be imported into malaria parasite and is also observed to bind to the 3’ UTR of Rad51 gene and P28 gene in Plasmodium berghei and Plasmodium falciparum respectively. The aim is to isolate PIP4K2a-specific nanobodies for further PIP4K2a pull down assays.

For selection and separation of PIP4K2a and PIP4K2a-specific yeast Nb, magnetic beads (MagneHis™ Protein Purification System) have been used as these are an ideal tool for immobilizing molecules like proteins or nucleic acids on a solid phase. These magnetic beads contain a magnetic core (typically ferromagnetic or supermagnetic) which is covered in usually agarose or silica which bind to proteins via affinity ligands. MagneHis™ Protein Purification System provides a simple, rapid, reliable method for the purification of polyhistidine- or HQ-tagged, expressed proteins. Paramagnetic precharged nickel particles (MagneHis™ Ni-Particles) are used to isolate polyhistidine- or HQ-tagged protein directly from a crude cell lysate using either a manual (requires use of a magnetic stand) or automated procedure. MagneHis™ Ni-Particles (magnetic beads) are compatible with a variety of common buffers and are suitable for use with culture volumes from 1ml–1 liter. Binding capacity is approximately 1 mg of polyhistidine-tagged protein per 1ml of MagneHis™ Ni-Particles.

magbeads2.png
    Diagram of MagneHis™ Protein Purification System Protocol.

    Objectives of the Research

    Overall objective

    The basic idea of this project is to isolate PIP4K2a-specific nanobodies from amongst a yeast surface display Nanobody library using magnetic beads and His-tagged purified PIP4K2a for separation and selection.

    METHODOLOGY

    Methods

    Dialysis

    Materials Required

    ·         150 mM NaCl, 10% Glycerol, 10 mM Tris, 1 mM EDTA, MiliQ water

    ·         Dialysis Bag

    ·         A few clips

    Dialysis Buffer (2L):
    Chemical Name Stock Concentration Working Concentration Volume Added (ml)
    NaCl 3 M 150 mM 100
    Glycerol 60% 10% 333.3
    Tris 1 M 10 mM 20
    EDTA 0.5 M 1 mM 4
    MiliQ water ── ── 1542.7

    Protocol

    1.      Prepared 2L of dialysis buffer and stored at 4°C.

    2.      Proper length of dialysis bag was cut and soaked in dialysis buffer.

    3.      The bottom of the dialysis bag was sealed well with a clip. Protein was added into the dialysis bag. The other end of the bag was also clipped.

    4.      A magnetic stirrer was put in the dialysis buffer and stirred at 4°C.

    5.      The dialysis buffer was changed after three hours and kept overnight.

    6.      The sample was collected from the dialysis bag; aliquots were made and stored at -20°C.

    Bradford assay

    Materials Required

    ·         5X Bradford Reagent, 70% Ethanol, MiliQ water, Cuvettes, Protein sample, Tissue paper

    ·         Spectrophotometer

    Protocol

    1.      In a cuvette, added 2 μl of dialysed protein, 798 μl of MiliQ water and 200 μl of Bradford Dye (final concentration of 1X).

    2.      Measured its absorbance at 595 nm.

    3.      Repeated for Undialysed protein.

    4.      Calculated the concentration of dialysed and undialysed protein using the following formula:

    x=[y(OD595)0.071/0.083]÷Reaction  volume(ml)\displaystyle x=\lbrack{y(OD595)–0.071}/0.083\rbrack\div Reaction\;volume(ml)

    Binding of His-tagged PIP4K2a with magnetic beads

    Materials Required

    ·         Magnetic beads, Wash buffer (dialysis buffer), Protein sample, Elution buffer

    ·         Magnetic Stand, Rotator

    Composition of elution buffer (1 ml)
    Reagents Stock Concentration Working Concentration Volume added (μl)
    572 3 M 150 mM 50
    Glycerol 60% 10% 166
    Tris 1 M 10 mM 10
    EDTA 0.5 M 1 mM 2
    Imidazole 1 M 200 mM 200
    GDW ── ── 572

     Protocol

    1.      Equilibrated 50 μl beads by suspending it in 1 ml of wash buffer (dialysis buffer) in an eppendorf and rotating at room temperature for 5 min. Repeated the washing step thrice.

    2.      Added 100 μl of protein sample to the washed beads.

    3.      Incubated at Room temperature for 2 minutes.

    4.      Put the Eppendorf on a magnetic stand and removed the unbound fraction. The unbound fraction (flow through) was collected separately.

    5.      Washed the protein bound beads with 1 ml wash buffer thrice.

    6.      Eluted the bound protein using 1 ml of elution buffer.

    7.      Loaded the flow through, Dialysed protein sample, eluate and beads on a gel to check for binding of protein to the beads.

    Nanobody Library recovery and expansion

    Materials Required

    ·         Yglc4.5 (-Trp) or SD media, Frozen Nanobody Library, 10% DMSO

    ·         Incubator, Spectrophotometer, Centrifuge machine, Water Bath

    Composition of Yglc4.5 (-Trp) Media (500 ml)
    Reagents Stock Concentration Working Concentration Volume/ Amount added
    Glucose 20% 2% 50 ml
    Leucine 10 X 1 X 5 ml
    Uracil 10 X 1 X 5 ml
    Histidine 10 X 1 X 5 ml
    Drop out media (-His, -Ura, -Leu, -Trp) 10 X 1 X 0.7 g
    Yeast Nitrogen Base (w/o amino acids and Ammonium sulphate) 10 X 1 X 0.85 g
    Ammonium Sulphate 10 X 1 X 2.5 g
    GDW ── ── 435 ml

     Protocol

    1.      Thawed an aliquot of frozen NbLib in a 30°C in a water bath.

    2.      Recovered the yeast in 1 L Yglc4.5 (-Trp) Media at 30°C overnight at 230 rpm.

    3.      Expanded to 3 L the next day and grew at 30°C, 230 rpm with shaking for 48 hours (usually OD should be between 10-30).

    4.      Measured OD600 to calculate density (1 OD= 1.5 x 107 yeast cells/ml).

    5.      Spinned down the cells at 5000 rpm for 5 min and resuspended in Yglc4.5 (-Trp) medium such that the final density became 1010 cells/ml.

    6.      Made aliquots in cryovials and stored at -20°C.  

    Induction of nanobody expression

    Materials Required

    ·         -Trp +Galactose medium, 10% DMSO, SD media

    ·         Incubator, cryovials

    Composition of -Trp +Galactose medium (250 ml)
    Reagents Stock Concentration Working Concentration Volume/ Amount added
    Galactose 20% 2% 25 ml
    Leucine 10 X 1 X 2.5 ml
    Uracil 10 X 1 X 2.5 ml
    Histidine 10 X 1 X 2.5 ml
    Drop out media (-His, -Ura, -Leu, -Trp) 10 X 1 X 0.35 g
    Yeast Nitrogen Base (w/o amino acids and Ammonium sulphate) 10 X 1 X 0.425 g
    Ammonium Sulphate 10 X 1 X 1.25 g
    GDW ── ── 217 ml

    Protocol

    1.      Spinned down the 3 L yeast culture.

    2.      Resuspended the pellet in SD media.

    3.      Removed 2 ml of culture for inoculation in 250 ml -Trp +Galactose medium. Incubated at 30°C, 230 rpm for 48 hours.

    4.      Made freezer stocks of the remaining culture using 10% DMSO and stored at -80°C.

    Nanobody selection

    Materials Required

    ·         Induced NbLib, wash buffer, selection buffer, elution buffer (800 μl SD media + 200 μl 1 M imidazole), magnetic beads, SD media, SD Agar, -Trp +Galactose medium, 10% DMSO, penstrep

    ·         Magnetic stand, Centrifuge machine, Rotator, Incubator, eppendorfs, falcon

    Composition of Selection buffer (100 ml)
    Reagents Stock Concentration Working Concentration Volume/ Amount added
    NaCl 3 M 150 mM 5 ml
    HEPES (pH 7.5) 0.89 M 20 mM 2.25 ml
    Maltose 1 M 5 mM 0.5 ml
    BSA ── 0.1% 0.1 g
    GDW ── ── 92.25 ml

    Protocol

    For Round I of selection:

    1.      Took 100 μl of beads and washed thrice with 1 ml of wash/dialysis buffer.

    2.      Added 500 μl of dialysed PIP4K2a and allowed binding for 1 min.

    3.      Put eppendorf on magnetic stand to remove the unbound protein fraction.

    4.      Washed the bound beads twice with wash buffer and once with selection buffer.

    5.      Spinned the induced yeast cells and resuspended the pellet in 10 ml chilled selection buffer in a falcon.

    6.      Added the PIP4K2a bound beads to the falcon and allowed binding in a rotator kept at 4°C for 1 hour.

    7.      Collected the flow through (unbound yeast cells) by placing in magnetic stand.

    8.      Washed the yeast-bound beads thrice with 1 ml selection buffer to remove non-specifically bound yeast cells.

    9.      Eluted the PIP4K2a-specific yeast cells in 1 ml elution buffer.

    10.  Plated 50 μl of the eluate in a SD Agar plate and incubated at 30°C for 48 hours.

    11.  Inoculated the remaining 950 μl eluate in 4 ml SD Media and incubated for 48 hours at 30°C.

    12.  Made freezer stocks of the flow through obtained and stored at -80°C.

    13.  Resuspended the colonies obtained on the plate (step 10) in 3 ml SD media.

    14.  Removed 0.5 ml from it for inoculation in 25 ml -Trp +Galactose medium. Incubated it at 30°C for 48 hours.

    15.  Made freezer stock of the remaining cells (in SD Media and 10% DMSO).

     For Round II of selection:

    1.      Repeated steps 1-9 from round I.

    2.      Diluted 50 μl of eluate to 20 times (50 μl in 950 μl SD Media) and plated 50 μl of it in a SD Agar plate.

    3.      Inoculated the remaining 950 μl eluate in 4 ml SD Media and incubated for 48 hours at 30°C.

    4.      Made freezer stocks of the flow through obtained and stored at -80°C.

    5.      Resuspended the colonies obtained on the plate in 3 ml SD media.

    6.      Removed 0.5 ml from it for inoculation in 25 ml -Trp +Galactose medium. Incubated it at 30°C for 48 hours.

    7.      Made freezer stock of the remaining cells (in SD Media and 10% DMSO).

     For Round III of selection:

    1.      Repeated steps 1-9 from round I.

    2.      Diluted 5 μl of eluate to 20 times (5 μl in 100 μl SD Media) and plated it in a SD Agar plate.

    3.      Made freezer stock of the flow through and the eluate left.

    Extraction of yeast plasmid (using glass beads)

    Materials Required

    ·         Solution I, Solution II, Solution III, TE Buffer, Phenol-chloroform-isoamyl alcohol (25:24:1), Isopropanol, 70% Ethanol, E. coli (DH5α) cells, glass beads, LB Agar, LB broth

    ·         Bead beater, water bath, ice, thermomixer

    Composition of Solution I (10 ml)
    Reagents Stock Concentration Working Concentration Volume added
    NaCl 3 M 100 mM 333 μl
    Tris HCl (pH 8.0) 1 M 10 mM 100 μl
    EDTA 0.5 M 1 mM 20 μl
    SDS 10% 0.1% 100 μl
    GDW ── ── 9.55 ml
    Composition of Solution II (10 ml)
    Reagents Stock Concentration Working Concentration Volume added
    NaOH 1 M 0.2 M 2 ml
    TritonX 100 % 10 % 1 ml
    GDW ── ── 7 ml

    Composition of Solution III (10 ml):

    3 M sodium acetate, pH 4.8 (dissolved 3.9 g in 8 ml GDW. Adjusted pH to 4.8 using glacial acetic acid. Readjusted volume to 10 ml using GDW).

    Composition of TE Buffer (10 ml)
    Reagents Stock Concentration Working Concentration Volume added
    Tris HCl 1 M 10 mM 100 μl
    EDTA (pH 8.0) 0.5 M 1 mM 20 μl
    GDW ── ── 9.88 ml

    Protocol

    • Spinned down yeast culture from 5 ml culture in 1.5 ml microcentrifuge tube.
    • Removed the media and washed the cells in 1 ml of water twice.
    • Re-suspended the yeast pellet in 200 μl of solution I.
    • Acid washed glass beads (0.5 mm diameter, autoclaved) were added until just below the level of the liquid and vortexed in bead beater for 2 min.
    • Ice cold solution II (200 μl) was then added and mixed by inversion.
    • 150 μl of ice cold solution III was added and the sample was again mixed thoroughly and incubated for 5 min in ice.
    • Equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added to the sample (still containing the glass beads) and this mixture was briefly vortexed and centrifuged for 2 min.
    • The aqueous upper phase was transferred to a second tube and the phenol chloroform extraction was repeated.
    • The aqueous layer was then transferred to a fresh tube and equal volume of isopropanol was added and incubated at -20°C for 30 min.
    • DNA was pelleted by centrifugation at 4°C for 10 min. This pellet was washed with 1 ml 70% ethanol (v/v) and carefully dried. Plasmid DNA was resuspended in 25 μl TE buffer.
    • The DNA was transformed into E. coli (DH5α).

    Extraction of yeast plasmid (using zymolyase)

    Materials Required

    ·         Potassium phosphate buffer (pH 7.5), EDTA (pH 8.0), 0.1% β-mercaptoethanol, Zymolyase, GDW

    ·         Weighing machine, Thermomixer

    Composition of potassium phosphate buffer (pH 7.5, 5.8 ml)
    Reagents Stock Concentration Working Concentration Volume/amount added
    K2HPO4 0.86 M 50 mM 0.87 g
    KH2PO4 0.86 M 50 mM 0.68 g
    Composition of zymolyase buffer (stock, 1 ml)
    Reagents Stock Concentration Working Concentration Volume/amount added
    Potassium phosphate buffer 0.86 M 50 mM 58 μl
    EDTA 0.5 M 5 mM 10 μl
    GDW ── ── 932 μl

     Zymolyase solution (50 μl):

    ·         Zymolyase buffer- 39.58 μl

    ·         Zymolyase- 2.5 mg (50 units)

    ·         β-mercaptoethanol- 10.42 μl

    Protocol

    1.      Picked up a PIP4K2a-specific yeast colony and grew in 1 ml SD media for 48 hours.

    2.      Spinned down the yeast culture and discarded the supernatant.

    3.      Washed the pellet with water once.

    4.      To the pellet added 50 μl of zymolyase solution and incubated at 30°C for 30 min in a thermomixer.

    Transformation of yeast plasmid DNA

    Materials Required

    ·         Competent E.coli (DH5α) cells, LB broth, LB Agar

    ·         Water bath (42°C), ice, Thermomixer, glass beads, incubator

    Protocol

    1.      Removed a tube of competent cells from -80°C freezer and thawed it on ice.

    2.      Added the transforming DNA (up to 25 ng per 50 μl of competent cells) in a volume not exceeding 5% of that of the competent cells. Mixed the contents of the tube and stored the tube on ice for 30 min.

    3.      Placed the tubes on a water bath (preheated to 42°C). Gave heat shock for exactly 90 sec.

    4.      Rapidly transferred the tubes to an ice bath. Allowed the cells to cool for 1-2 min.

    5.      Added 800 μl of LB broth to the tube and transferred it to a shaking thermomixer set at 37°C. Incubated for 45 min.

    6.      Plated 100 μl on a LB plate containing the appropriate antibiotic and incubated at 37°C.

    Plasmid isolation from E.coli (DH5α) cells

    Materials Required

    ·         APS Labs Plasmid Miniprep Purification Kit (cat#: MAGSPIN-02)

    ·         Spin columns, microcentrifuge  

    Protocol

    1.      Pelleted 1-10 ml of bacteria culture by centrifugation for 1 min at top speed (12-14,000x g) in a microcentrifuge. Discarded the supernatant and removed any excess media.

    2.      Resuspended the cell pellet completely in 200 μl of Solution I by pipetting.

    3.      Added 200 μl of Solution II and mixed by inverting the tube 5 times.

    4.      Added 200 μl of Solution III and mixed by inverting the tube 5 times.

    5.      Centrifuged the lysate at top speed in a microcentrifuge for 5 min.

    6.      Inserted the spin column into a collection tube, carefully transferred all of the clear lysate from step 5 to spin column; centrifuged at top speed for 1 min.

    7.      Discarded the filtrate from the collection tube and added 700 μl of Wash solution to the spin column and centrifuged at top speed for 1 min. Repeated this step once more.

    8.      Discarded the filtrate and centrifuged at top speed for additional 3-5 min to remove residual trace of ethanol.

    9.      Transferred the spin column into a new microcentrifuge tube and added 50-100 μl of Elution solution into the column and waited for 1-2 min.

    10.  Centrifuged at top speed for 1 min to elute the DNA. Stored the eluted plasmid DNA at -20°C.

    PCR of yeast plasmid

    Materials Required

    • dNTP mix, forward primer, reverse primer, GDW, Pfu DNA Polymerase, Pfu buffer, MgSO4
    • Thermal cycler, ice

    Protocol

    1. In a sterile, nuclease-free microcentrifuge tube, combined the following components:

    2. Immediately placed the reactions in a thermal cycler that was preheated to 95°C .

    3. Started the thermal cycling program.

    Thermal cycling conditions for Pfu DNA Polymerase-mediated PCR amplification
     StepTemperature Time Number of cycles 
     Initial Denaturation95°C 5 min 1 cycle 
     Denaturation I 95°C30 sec 5 cycles  
     Annealing I  50°C30 sec 
     Extension I 72°C1 min 
     Denaturation II 95°C30 sec     25 cycles  
     Annealing II 60°C30 sec   
     Extension II 72°C1 min  
    Final Extension 72°C 10 min 1 cycle 
     Storage 4°C Indefinite1 cycle 

    RESULTS AND DISCUSSION

    Analysis of Dialysed and Undialysed Protein through Bradford Assay

    The protein was dialysed to remove imidazole present in its buffer for efficient binding to magnetic beads. Bradford assay was performed for both dialysed as well as undialysed protein (PIP4K2a) to check for the concentration and factor change in dilution of protein in both the cases.

    Concentration of undialysed protein= 0.79 μg/μl (for 5 μg of protein, 6.32 μl of protein was loaded)

    Concentration of dialysed protein= 0.55 μg/μl

    Dilution factor= 0.79/0.55 = 1.43

    The fold change in dilution was further confirmed by loading the same volume (6.32 μl) of both the protein samples for SDS-PAGE.

    3_1.png
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    SDS-PAGE of dialysed and undialysed PIP4K2a

    Binding of His-tagged PIP4K2a with Magnetic Beads

    The dialysed protein was allowed to bind to the magnetic beads and the flow through, eluate and beads were loaded on a polyacrylamide gel to check for the presence of protein in each of them.

    4.png
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    Result obtained through SDS-PAGE confirms that PIP4K2a binds to the magnetic beads used in this experiment.

    Library Recovery, Expansion and Induction

    Expanded yeast NbLib to 3 L and measured OD600 after 48 hours. A density of 1.6 x 1011 cells/ 3 L was obtained. Cells were induced to express nanobodies by resuspending them in -Trp Galactose media.

    Nanobody Selection

    Three rounds of nanobody selection were performed to get yeast colonies expressing nanobodies with more specificity to PIP4K2a. In each successive round, their was an increase in the number of colonies obtained, pertaining to the increased specificity of the yeast Nb to PIP4K2a.

    6_1.png
      PIP4K2a-specific yeast colonies obtained in 3 rounds of nanobody selection

      Extraction and Transformation of Yeast Plasmid

      Plasmids were isolated from yeast colonies (obtained in round 3 of nanobody selection) using glass beads and transformed into E.coli (DH5α) cells for cloning. Three transformed colonies were obtained from three different yeast colonies picked up. Plasmids were again isolated from these transformed colonies and amplified by PCR. Plasmids were also isolated from yeast colonies using zymolyase treatment and amplified using PCR.

      Amplification of Nb Plasmid

      Plasmids obtained using both glass beads as well as zymolyase method were amplified in a PCR reaction and run on an agarose gel to check for the presence and size of amplicon (to confirm whether the appropriate Nb gene has been amplified).

      Unfortunately, no PCR amplicon of appropriate size (~400-500 bp) could be seen on the agarose gel after visualisation. An appropriate result for the positive control was also not obtained (suggesting that maybe the primers used in the experiment were faulty).

      Direct PCR of cell lysate using zymolase treatment as well as of plasmid obtained using glass bead extraction was also performed and run on agarose gel. No appropriate PCR amplicon could be visualised in the above case too.

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      Agarose gel electrophoresis of Nb plasmid-PCR product (after glass bead extraction)
      9.png
      Agarose Gel Electrophoresis of Nb plasmid- PCR product (after zymolyase treatment)
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      Agarose Gel Electrophoresis of plasmids obtained from transformed colonies
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      CONCLUSION

      In this entire experiment, I was successfully able to grow yeast cells, recover and expand yeast NbLib as well as screen for PIP4K2a-specific yeast nanobody from amongst the yeast NbLib. Outcome of further steps performed could not be verified due to some fault in the PCR reaction mixture.

      With proper PCR reaction mix and conditions, the appropriate Nb gene fragment can be amplified, cloned into E.coli cells and used for PIP4K2a pull-down assys in the future.

      REFERENCES

      • Hamers-Casterman C, et al. Naturally occurring antibodies devoid of light chains. Nature. 1993;363:446–448. doi: 10.1038/363446a0.
      • Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev.
        Biochem. 82, 775–797 (2013).
      • McMahon, C. Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nature structural and molecular biology. 25, 289-296 (2018).
      • Uchańsk, T. An improved yeast surface display platform for the screening of nanobody immune libraries. Scientific Reports. 9:382 (2019).

      ACKNOWLEDGEMENTS

      I would like to take this opportunity to thank Indian Academy of Sciences for providing me the opportunity to work under the Summer Research fellowship programme in National Center for Cell Science, Pune. I extend my heartful gratitude to Dr. Vasudevan Seshadri, for providing me the opportunity to work under his supervision. His support and valuable suggestions have played a big role in shaping this project work. I would also like to express my sincere thanks to the lab members for always being there to guide me with their knowledge and helping me to learn an array of techniques in this duration.

      Source

      • Fig 1: https://www.promega.in/products/protein-purification/protein-purification-kits/magnehis-protein-purification-system/?catNum=V8500
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