WO2011074941A1 - A method of high throughput screening of inhibitors that inhibit interaction of an attachment protein to cell receptors using recombinant phage display - Google Patents

A method of high throughput screening of inhibitors that inhibit interaction of an attachment protein to cell receptors using recombinant phage display Download PDF

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Publication number
WO2011074941A1
WO2011074941A1 PCT/MY2010/000322 MY2010000322W WO2011074941A1 WO 2011074941 A1 WO2011074941 A1 WO 2011074941A1 MY 2010000322 W MY2010000322 W MY 2010000322W WO 2011074941 A1 WO2011074941 A1 WO 2011074941A1
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Prior art keywords
recombinant protein
phage
target polypeptides
bacterial cells
infected
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PCT/MY2010/000322
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French (fr)
Inventor
Abu Bakar Sazaly
Abd-Jamil Juraina
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Universiti Malaya
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Publication of WO2011074941A1 publication Critical patent/WO2011074941A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Definitions

  • the present invention relates to a modified process or method to be used for high throughput screening (hereinafter referred to as "HTS”) of molecules both peptides and non-peptides that inhibit binding of an attachment protein displayed on recombinant phage.
  • HTS high throughput screening
  • Phage display or related applications are widely used for the study of peptide-peptide or peptide-DNA interaction. It has been used for high-throughput screening of protein interaction especially in the determination of antigen-antibody reaction in between pol3peptides. From the principal concept of this approach, various modifications have been made thereon to improve its applicability.
  • United States patent 2005/0014135 Al discloses a method for the selection and identification from a plurality of peptide or protein molecules which can specifically interact with at least one representative from a plurality of target molecules, forming a bond.
  • the inventive method comprises of a) a virus system consisting of a plurality of viruses, wherein each virus respectively presents at least on representative from the plurality of peptide or protein molecules on the surface thereof, is brought into contact with the plurality of target molecules which are immobilized on the surface of a solid phase carrier such that they are positioned addressable in a two-dimensional grade; b) unbound viruses removed from the surface thereof; and c) the interaction partner is identified by detection and determination of the position of the bond between the immobilized" ligand and the interaction partner presented by the virus with the marker- free detection method.
  • our invention is a modified method of the existing invention to be used for high throughput screening of molecules that bind to or inhibit binding of an attachment protein displayed on phage.
  • the present invention also differs from the previous disclosure in that it uses the gene of a specific attachment protein to be displayed on the phage.
  • the present invention can be used for the selection of protein/peptide molecules that specifically bind to the displayed attachment protein and to identify potential molecules that can inhibit binding of the attachment protein following pre-incubation of the displayed attachment protein with molecules that may act as inhibitors.
  • the United States patent US 6,589,730 B l discloses an invention that relates to the screening for protein-protein interactions, and in particular, to identification of specific protein-protein interaction which lead to internalization of the protein-protein complex and transgene expression in the target cell.
  • the invention comprises of a) contacting one or more ligand displaying genetic package(s) with a cell(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package, and wherein the cell(s) expresses an anti-ligand receptor fusion protein on its surface; b) detecting product expressed by the cell(s), and c) recovering a nucleic acid molecule encoding an internalizing ligand and/or a nucleic acid molecule encoding an internalizing anti-ligand from the cell(s) expressing the product, thereby identifying a ligand or anti-ligand pairs of an internalizing ligand/anti-ligand pair.
  • the present process/method relates to the use of gene-targeted phage display for high throughput screening of molecules that bind to or inhibit binding of the displayed attachment protein on phage. It exempts the use of a cell culture system and introduction of an anti-ligand or ligand is performed through its adherence in a 96-well plate by ionic or charge interaction.
  • the patent WO0118234 (Al) relates to a method of monitoring ligand/receptor interaction through the use of phage display where the ligand is a typical small pharmaceutical molecule.
  • the invention entails a) the coupling of a small molecule onto a solid support, preferably but not necessarily through a coupling system that is subject to elution and using reagents which are independent of the nature of the small molecule; b) the incorporation of genes encoding non-viral proteins into the viral genome so as to result in the production of fusion proteins between the desired foreign proteins and a coat protein of the virus, whereby when the virus replicates, the foreign protein is displayed at its surface; c) the library of displayed proteins is then contacted with "bait" bound to the solid support; d) the successfully interacting displayed proteins will then preferentially be retained on the solid support and the non-binding proteins washed away; e) the bound protein-displaying phage are then eluted and amplified by cul
  • invention WOOl 18234 uses "baits" or ligands consisting only « of small molecules such as synthetic natural products, drugs and polyketides, which are not proteins or peptides.
  • invention WOOl 18234 makes, use of support such as agarose, polystyrene or other polyvinyl compounds, and magnetic beads that require their installation in an affinity column.
  • the present invention aims to provide a method for high throughput screening (HTS) of molecules capable in preventing polypeptide-polypeptide interaction, where the molecules can be a chemical compound, protein or even glyco-peptides.
  • HTS high throughput screening
  • Another object of the present invention is to offer a simple method to evaluate binding affinity of a molecule and/or polypeptides towards a targeted protein.
  • one of the embodiments of the present invention includes a method of identifying a potential reagent that inhibits reaction between a target polypeptide and a recombinant protein comprising the steps of providing a plurality of target polypeptides attached on a solid platform; bringing a potential reagent into contact with and bind onto a plurality of recombinant protein expressed on a phage display system: bringing the recombinant protein into contact with the target polypeptides; removing unbound recombinant protein and the phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected host cells; whereby binding of the potential reagent onto the recombinant protein inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential rea
  • the steps of the method may comprise of providing a plurality of target polypeptides attached on a solid platform; bringing a plurality of a potential reagent into contact with the target polypeptides; removing unbound potential reagents; adding a plurality of recombinant protein. on a phage .
  • the display system into contact with the target polypeptides; removing unbound recombinant protein; adding a plurality of bacterial host cells capable of being infected by the phage to the solid platform; and determining the presence and number of infected bacterial cells; whereby binding of the potential reagent onto the target polypeptides inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the target polypeptides allows subsequent infection of the bacterial cells by the recombinant phage.
  • the competency of the potential reagent to inhibit the target polypeptides in comparison to the recombinant protein can be determined.
  • Further aspect to determine if the recombinant protein is capable of binding to a target polypeptide on a solid surface includes providing a plurality of a target polypeptide attached on a solid platform; bringing a plurality of a recombinant protein expressed on a phage display system into contact with the target polypeptide; removing unbound recombinant protein and phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected bacterial host cells; whereby capability of the recombinant protein to bind to the target protein allows subsequent infection of the bacterial cells by the recombinant phage.
  • the capability of the recombinant protein to interact with the target protein can be determined; whereby the target protein can be any protein or polypeptide of a homogenous mixture and purity.
  • the disclosed method is able to identify the interaction between a potential reagent and a recombinant protein expressed on a phage display system and determine binding inhibition of the reagent against the recombinant protein to target polypeptides where the target polypeptides are lysate of cells containing polypeptides of different conformation.
  • the step of determining the presence or number of the infected bacterial cells is conducted through plating of the infected bacterial .cells, on a. suitable medium followed by the counting colonies of produced from the plated bacterial cells.
  • Figure 1 shows the map of different region of DENV-2 E displayed
  • Figure 2 is a gel picture showing different amplified fragments of DENV-2 E in which Lane 1 is 100 bp plus DNA marker, lane 2 - 7 are the different amplified regions of DENV-2 E;
  • Figure 3 is an SDS-PAGE gel showing the separated EN, EB, EB2, EB3, EB4, and EB5 in lane 2 to 7 as well as a pre-stained protein marker in Lane 1 ; and Figure 4 includes graphs showing the binding power of the truncated DENV-2
  • the disclosed method of identifying a potential reagent to inhibit interaction between target polypeptides and a recombinant protein expressed on a phage display system comprises the steps of providing a plurality of target polypeptides attached on a solid platform; bringing a potential reagent into contact with and bind onto the recombinant protein: bringing a plurality of the recombinant protein , into contact with the target polypeptides; removing unbound recombinant protein and the phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected host cells; whereby binding of the potential reagent onto the recombinant protein inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while the incapability of the potential reagent to bind onto the recombinant protein allows subsequent infection of the bacterial cells by the recombinant
  • the present invention provides a modified process or method to be used for the purpose of HTS of molecules or natural and synthetic compounds that bind to or inhibit binding of an attachment protein displayed on phage to their targets or receptors that are coated onto the solid platform allowing for high throughput screening.
  • the solid platform is microtiter plates.
  • the coating of the plurality of target polypeptides is done by incubating the target polypeptides in 0.05 M sodium bicarbonate, pH 9.6 in a 96- well plate overnight at 4 °C or 2 hours at 37 °C.
  • the target polypeptides in fact, are derived from lysate of cells that contain polypeptides of different conformations.
  • the target polypeptides can also be a plurality of proteins tested for potential interaction with the recombinant protein.
  • One skilled in the art shall appreciate the fact that modification may be made to the above mentioned parameters owing to the target polypeptides or recombinant protein used, and such modification shall not depart from the scope of the present invention.
  • the potential reagent to be tested for its capability to inhibit or interfere binding of the recombinant protein can be a chemical compound, polypeptides, glycoprotein or small compounds.
  • the recombinant polypeptide is expressed on a phage display system through any known method in the field.
  • the selected recombinant protein gene is amplified by polymerase chain reaction (PCR) and the amplified fragment is purified for subsequent cloning.
  • PCR polymerase chain reaction
  • Both the vector or phagemid and the amplified fragment were excised with suitable restriction enzymes.
  • the latter is ligated into the phagemid and the recombinant construct is transformed into a suitable prokaryotic host using the calcium chloride method.
  • the display of the recombinant protein is performed by infecting the recombinant bacteria with M13 helper phage.
  • the recombinant phage displaying the recombinant protein and containing the recombinant phagemid material were precipitated using polyethylene glycol/sodium chloride mixture for 30 minutes on ice after which the precipitated phage were sedimented at 20,000 g and recovered.
  • the recombinant phage was serially diluted and mixed with bacterial cells to infect the bacterial cells with the phage.
  • the bacterial cells were spread on a selective medium agar and colonies that grow on the selective medium agar were counted to determine the amount or titer of recombinant phage displaying the recombinant polypeptide. More preferably, the bacterial cells are E.coli TG1 and the phage system is M13 helper phage.
  • the present invention may be embodied in another aspect in which the method comprises the steps of providing a plurality of target polypeptides attached on a solid platform; bringing a plurality of a potential reagent or inhibitor and a plurality of a recombinant protein expressed on a phage display system together into contact as well as to bind them onto the target polypeptides; removing the unbound potential reagent and recombinant protein; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected bacterial cells; whereby binding of the potential reagent to the recombinant protein inhibits interaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the recombinant protein allows subsequent infection of the bacterial cells by the recombinant phage displaying the recombinant protein.
  • the steps may include providing a plurality of target polypeptides attached on a solid platform; bringing a plurality of a potential reagent into contact to the target polypeptides; removing the unbound reagent; adding a plurality of recombinant protein expressed on a phage display system into contact with the target polypeptides; removing unbound recombinant protein; adding a plurality of bacterial host cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected host cells; whereby binding of the potential reagent onto the target polypeptides inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the target polypeptides allows subsequent infection of the bacterial cells by the recombinant phage.
  • the competency of the potential reagent to inhibit binding of the recombinant protein to the target polypeptides may include providing a
  • the method may include the steps of providing a plurality of a target polypeptide attached on a solid platform; bringing a plurality of a recombinant protein expressed on a phage display system into contact with the target polypeptide; removing unbound recombinant protein and phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected bacterial host cells: whereby capability of the recombinant protein to bind to the specific target polypeptide allows subsequent infection of the bacterial cells by the recombinant phage. Following this method.- the capability of the recombinant protein to interact with the target polypeptide can be determined; whereby the specific target protein can be any protein or polypeptide of a homogenous mixture and purity.
  • the step determining the presence or number of the infected bacterial host cells is conducted through the plating of the infected bacterial cells onto a suitable medium followed by the counting of colonies produced from the plated bacterial cells.
  • successful binding of the potential reagent to the recombinant protein or to the target polypeptides shall prohibit interaction between the recombinant polypeptide and target polypeptides.
  • the presence of the bacterial cell colonies indicates successful binding of the recombinant protein onto the target polypeptides while the potential reagent is known to be less effective in terms of binding affinity to the recombinant protein or inhibiting interaction between the recombinant protein and target polypeptides.
  • the present invention is exemplified here by using the attachment protein of dengue virus type 2 (DENV-2), which is the envelope protein (E).
  • DENV-2 dengue virus type 2
  • E envelope protein
  • the envelope protein was amplified from the harvested RNA of DENV-2 infected cells using specific primers and cloned into pGEM-T cloning vector.
  • the E gene fragment from pGEMT-T was then excised and inserted into phagemid pCANTAB-5E using the available engineered restriction sites.
  • the recombinant phagemid was transformed into E. coli TGI to display on phage.
  • telomeres were grown in growing medium 2xYT and expression was induced using 1 mM isopropyl-B-D-thiogalactopyranoside (TPTG) for four hours.
  • TPTG isopropyl-B-D-thiogalactopyranoside
  • the TGI culture was then clarified by centrifugation and SDS reducing sample buffer was added to lyse the E. coli pellet and prepare the proteins for SDS- PAGE separation. Proteins that were separated were transferred onto an Immobilon- NC membrane for western blotting.
  • Detection of recombinant E was performed using antibodies specific against the Flag tag engineered at the C-terminus of the recombinant E protein, and using antibodies specific against the phage.
  • the HTS binding and inhibition assay was performed on cell protein lysate.
  • the lysate (1-3 ug) was previously coated in 96-well test plates overnight at 4 °C using 0.05 M sodium carbonate (Na 2 C0 3 ). ph 9.6.
  • Coated cell lysate was blocked with 1% polyvinyl-pyrrolidone (PVP) containing 0.02% tween detergent for two hours at room temperature.
  • PVP polyvinyl-pyrrolidone
  • CDRL2-SS or CDRH3-SS were added into the recombinant phage mix ( ⁇ 5 x 10 6 particles). They were incubated for two hours at 37 °C.
  • the phage mix was added into the test plates and the test plates were incubated at 37 °C for one hour. Unbound phage were removed using PBS containing 0.05% Tween detergent. Phage that bound to the coated cell lysate was saved by adding TGI E. coli into the test plates and incubating the plate at 37 °C for 30 minutes. The E. coli was then spread on a selective medium agar and the number of colonies was determined the following day. The number of colonies was representative of the number of recombinant phage that bound to the cell protein lysate.

Abstract

A method of identifying a potential reagent to inhibit reaction between target polypeptides and a recombinant protein comprises the steps of providing a plurality of target polypeptides attached on a solid platform; bringing a plurality of a potential reagent into contact with and bind onto the recombinant protein that is expressed and displayed on phage; bringing the plurality of the recombinant protein and potential reagent into contact with target polypeptides; removing unbound recombinant protein and phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected bacterial cells; whereby binding of the potential reagent onto the recombinant protein inhibits binding or interaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the recombinant protein allows subsequent infection of the bacterial cells by the recombinant phage.

Description

A METHOD OF HIGH THROUGHPUT SCREENING OF INHIBITORS THAT INHIBIT INTERACTION OF AN ATTACHMENT PROTEIN TO CELL RECEPTORS USING RECOMBINANT PHAGE DISPLAY FIELD OF THE INVENTION
The present invention relates to a modified process or method to be used for high throughput screening (hereinafter referred to as "HTS") of molecules both peptides and non-peptides that inhibit binding of an attachment protein displayed on recombinant phage.
BACKGOUND OF THE INVENTION
Phage display or related applications are widely used for the study of peptide-peptide or peptide-DNA interaction. It has been used for high-throughput screening of protein interaction especially in the determination of antigen-antibody reaction in between pol3peptides. From the principal concept of this approach, various modifications have been made thereon to improve its applicability. For example, United States patent 2005/0014135 Al discloses a method for the selection and identification from a plurality of peptide or protein molecules which can specifically interact with at least one representative from a plurality of target molecules, forming a bond. The inventive method comprises of a) a virus system consisting of a plurality of viruses, wherein each virus respectively presents at least on representative from the plurality of peptide or protein molecules on the surface thereof, is brought into contact with the plurality of target molecules which are immobilized on the surface of a solid phase carrier such that they are positioned addressable in a two-dimensional grade; b) unbound viruses removed from the surface thereof; and c) the interaction partner is identified by detection and determination of the position of the bond between the immobilized" ligand and the interaction partner presented by the virus with the marker- free detection method. Whereby the method makes it possible to concentrate viruses presenting interaction partners by means of an optionally cyclic repetition of selection, and the selected interaction partners are recombinantly expressed after identification of the coding nucleotide sequences, our invention is a modified method of the existing invention to be used for high throughput screening of molecules that bind to or inhibit binding of an attachment protein displayed on phage. The present invention also differs from the previous disclosure in that it uses the gene of a specific attachment protein to be displayed on the phage. The present invention can be used for the selection of protein/peptide molecules that specifically bind to the displayed attachment protein and to identify potential molecules that can inhibit binding of the attachment protein following pre-incubation of the displayed attachment protein with molecules that may act as inhibitors.
The United States patent US 6,589,730 B l discloses an invention that relates to the screening for protein-protein interactions, and in particular, to identification of specific protein-protein interaction which lead to internalization of the protein-protein complex and transgene expression in the target cell. The invention comprises of a) contacting one or more ligand displaying genetic package(s) with a cell(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package, and wherein the cell(s) expresses an anti-ligand receptor fusion protein on its surface; b) detecting product expressed by the cell(s), and c) recovering a nucleic acid molecule encoding an internalizing ligand and/or a nucleic acid molecule encoding an internalizing anti-ligand from the cell(s) expressing the product, thereby identifying a ligand or anti-ligand pairs of an internalizing ligand/anti-ligand pair. Whereas the invention/method is providing a method of identifying a ligand or anti-ligand of an internalizing ligand/anti-ligand interactions, the present process/method relates to the use of gene-targeted phage display for high throughput screening of molecules that bind to or inhibit binding of the displayed attachment protein on phage. It exempts the use of a cell culture system and introduction of an anti-ligand or ligand is performed through its adherence in a 96-well plate by ionic or charge interaction.
The patent WO0118234 (Al) relates to a method of monitoring ligand/receptor interaction through the use of phage display where the ligand is a typical small pharmaceutical molecule. . The invention entails a) the coupling of a small molecule onto a solid support, preferably but not necessarily through a coupling system that is subject to elution and using reagents which are independent of the nature of the small molecule; b) the incorporation of genes encoding non-viral proteins into the viral genome so as to result in the production of fusion proteins between the desired foreign proteins and a coat protein of the virus, whereby when the virus replicates, the foreign protein is displayed at its surface; c) the library of displayed proteins is then contacted with "bait" bound to the solid support; d) the successfully interacting displayed proteins will then preferentially be retained on the solid support and the non-binding proteins washed away; e) the bound protein-displaying phage are then eluted and amplified by culruring them in cells to produce a library enriched in proteins which bind the bait; and f) the nucleotide sequences contained in the genome of these phage can then be sequenced and the amino acid sequence of the binding protein deduced. Whereas the presently disclosed invention uses a similar method of using a solid support for the coupling of ligands or anti-ligands, invention WOOl 18234 (Al) uses "baits" or ligands consisting only« of small molecules such as synthetic natural products, drugs and polyketides, which are not proteins or peptides. Unlike the. presently disclosed invention which uses 96 well plates as the solid support and where the interaction is ionic or charge based, invention WOOl 18234 (Al) makes, use of support such as agarose, polystyrene or other polyvinyl compounds, and magnetic beads that require their installation in an affinity column. SUMMARY OF THE INVENTION
The present invention aims to provide a method for high throughput screening (HTS) of molecules capable in preventing polypeptide-polypeptide interaction, where the molecules can be a chemical compound, protein or even glyco-peptides. Another object of the present invention is to offer a simple method to evaluate binding affinity of a molecule and/or polypeptides towards a targeted protein.
At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention includes a method of identifying a potential reagent that inhibits reaction between a target polypeptide and a recombinant protein comprising the steps of providing a plurality of target polypeptides attached on a solid platform; bringing a potential reagent into contact with and bind onto a plurality of recombinant protein expressed on a phage display system: bringing the recombinant protein into contact with the target polypeptides; removing unbound recombinant protein and the phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected host cells; whereby binding of the potential reagent onto the recombinant protein inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the recombinant protein allows subsequent infection of the bacterial cells by the recombinant phage.
In another aspect of the present invention, the steps of the method may comprise of providing a plurality of target polypeptides attached on a solid platform; bringing a plurality of a potential reagent into contact with the target polypeptides; removing unbound potential reagents; adding a plurality of recombinant protein. on a phage . display system into contact with the target polypeptides; removing unbound recombinant protein; adding a plurality of bacterial host cells capable of being infected by the phage to the solid platform; and determining the presence and number of infected bacterial cells; whereby binding of the potential reagent onto the target polypeptides inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the target polypeptides allows subsequent infection of the bacterial cells by the recombinant phage. Through this embodiment, the competency of the potential reagent to inhibit the target polypeptides in comparison to the recombinant protein can be determined.
Further aspect to determine if the recombinant protein is capable of binding to a target polypeptide on a solid surface includes providing a plurality of a target polypeptide attached on a solid platform; bringing a plurality of a recombinant protein expressed on a phage display system into contact with the target polypeptide; removing unbound recombinant protein and phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected bacterial host cells; whereby capability of the recombinant protein to bind to the target protein allows subsequent infection of the bacterial cells by the recombinant phage. Following this method, the capability of the recombinant protein to interact with the target protein can be determined; whereby the target protein can be any protein or polypeptide of a homogenous mixture and purity.
The disclosed method is able to identify the interaction between a potential reagent and a recombinant protein expressed on a phage display system and determine binding inhibition of the reagent against the recombinant protein to target polypeptides where the target polypeptides are lysate of cells containing polypeptides of different conformation.
According to another aspect, the step of determining the presence or number of the infected bacterial cells is conducted through plating of the infected bacterial .cells, on a. suitable medium followed by the counting colonies of produced from the plated bacterial cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the map of different region of DENV-2 E displayed
phage;
Figure 2 is a gel picture showing different amplified fragments of DENV-2 E in which Lane 1 is 100 bp plus DNA marker, lane 2 - 7 are the different amplified regions of DENV-2 E;
Figure 3 is an SDS-PAGE gel showing the separated EN, EB, EB2, EB3, EB4, and EB5 in lane 2 to 7 as well as a pre-stained protein marker in Lane 1 ; and Figure 4 includes graphs showing the binding power of the truncated DENV-2
E fragments displayed on Ml 3 phages towards different cell lines in cell-binding studies to C6/36, Vero and THP- l cells (a) and inhibition of binding of the recombinant protein EB4 to cell lysate with the reagent CDRL2-SS and CDRH 3-SS (b).
DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that the present invention may be embodied in other specific forms and is not limited to the sole embodiment described above. However, modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto.
According to the preferred embodiment, the disclosed method of identifying a potential reagent to inhibit interaction between target polypeptides and a recombinant protein expressed on a phage display system comprises the steps of providing a plurality of target polypeptides attached on a solid platform; bringing a potential reagent into contact with and bind onto the recombinant protein: bringing a plurality of the recombinant protein , into contact with the target polypeptides; removing unbound recombinant protein and the phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected host cells; whereby binding of the potential reagent onto the recombinant protein inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while the incapability of the potential reagent to bind onto the recombinant protein allows subsequent infection of the bacterial cells by the recombinant phage. The present invention provides a modified process or method to be used for the purpose of HTS of molecules or natural and synthetic compounds that bind to or inhibit binding of an attachment protein displayed on phage to their targets or receptors that are coated onto the solid platform allowing for high throughput screening. Preferably, the solid platform, but not limited to, is microtiter plates. In more specific, the coating of the plurality of target polypeptides is done by incubating the target polypeptides in 0.05 M sodium bicarbonate, pH 9.6 in a 96- well plate overnight at 4 °C or 2 hours at 37 °C. In the most preferred embodiment, the target polypeptides in fact, are derived from lysate of cells that contain polypeptides of different conformations. The target polypeptides can also be a plurality of proteins tested for potential interaction with the recombinant protein. One skilled in the art shall appreciate the fact that modification may be made to the above mentioned parameters owing to the target polypeptides or recombinant protein used, and such modification shall not depart from the scope of the present invention.
In respect to the preferred embodiment, the potential reagent to be tested for its capability to inhibit or interfere binding of the recombinant protein can be a chemical compound, polypeptides, glycoprotein or small compounds.
Preferably, the recombinant polypeptide is expressed on a phage display system through any known method in the field. For example, the selected recombinant protein gene is amplified by polymerase chain reaction (PCR) and the amplified fragment is purified for subsequent cloning. Both the vector or phagemid and the amplified fragment were excised with suitable restriction enzymes. The latter is ligated into the phagemid and the recombinant construct is transformed into a suitable prokaryotic host using the calcium chloride method. The display of the recombinant protein is performed by infecting the recombinant bacteria with M13 helper phage. Following the infection, the recombinant phage displaying the recombinant protein and containing the recombinant phagemid material (recombinant phage) were precipitated using polyethylene glycol/sodium chloride mixture for 30 minutes on ice after which the precipitated phage were sedimented at 20,000 g and recovered. As set forth, to determine the titer of the recombinant phage expressing the recombinant protein, the recombinant phage was serially diluted and mixed with bacterial cells to infect the bacterial cells with the phage. The bacterial cells were spread on a selective medium agar and colonies that grow on the selective medium agar were counted to determine the amount or titer of recombinant phage displaying the recombinant polypeptide. More preferably, the bacterial cells are E.coli TG1 and the phage system is M13 helper phage. It is important to note that the present invention may be embodied in another aspect in which the method comprises the steps of providing a plurality of target polypeptides attached on a solid platform; bringing a plurality of a potential reagent or inhibitor and a plurality of a recombinant protein expressed on a phage display system together into contact as well as to bind them onto the target polypeptides; removing the unbound potential reagent and recombinant protein; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected bacterial cells; whereby binding of the potential reagent to the recombinant protein inhibits interaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the recombinant protein allows subsequent infection of the bacterial cells by the recombinant phage displaying the recombinant protein. Through this embodiment, the capacity of the potential reagent to inhibit the binding of the recombinant protein to the target polypeptides can be determined.
In a further embodiment, the steps may include providing a plurality of target polypeptides attached on a solid platform; bringing a plurality of a potential reagent into contact to the target polypeptides; removing the unbound reagent; adding a plurality of recombinant protein expressed on a phage display system into contact with the target polypeptides; removing unbound recombinant protein; adding a plurality of bacterial host cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected host cells; whereby binding of the potential reagent onto the target polypeptides inhibits reaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the target polypeptides allows subsequent infection of the bacterial cells by the recombinant phage. Through this embodiment, the competency of the potential reagent to inhibit binding of the recombinant protein to the target polypeptides can be determined.
In a further embodiment, the method may include the steps of providing a plurality of a target polypeptide attached on a solid platform; bringing a plurality of a recombinant protein expressed on a phage display system into contact with the target polypeptide; removing unbound recombinant protein and phage; adding a plurality of bacterial cells capable of being infected by the phage to the solid platform; and determining the presence or number of the infected bacterial host cells: whereby capability of the recombinant protein to bind to the specific target polypeptide allows subsequent infection of the bacterial cells by the recombinant phage. Following this method.- the capability of the recombinant protein to interact with the target polypeptide can be determined; whereby the specific target protein can be any protein or polypeptide of a homogenous mixture and purity.
In accordance with the preferred embodiment, the step determining the presence or number of the infected bacterial host cells is conducted through the plating of the infected bacterial cells onto a suitable medium followed by the counting of colonies produced from the plated bacterial cells. Pursuant to the preferred embodiment, successful binding of the potential reagent to the recombinant protein or to the target polypeptides shall prohibit interaction between the recombinant polypeptide and target polypeptides. Therefore, in the embodiment where the recombinant protein is expressed on a phage display system, the presence of the bacterial cell colonies indicates successful binding of the recombinant protein onto the target polypeptides while the potential reagent is known to be less effective in terms of binding affinity to the recombinant protein or inhibiting interaction between the recombinant protein and target polypeptides.
The following example is intended to further illustrate the invention, without any intent for the invention to be limited to the specific embodiments described therein.
Example 1
The present invention is exemplified here by using the attachment protein of dengue virus type 2 (DENV-2), which is the envelope protein (E). The envelope protein was amplified from the harvested RNA of DENV-2 infected cells using specific primers and cloned into pGEM-T cloning vector. The E gene fragment from pGEMT-T was then excised and inserted into phagemid pCANTAB-5E using the available engineered restriction sites. The recombinant phagemid was transformed into E. coli TGI to display on phage. To assay for successful expression and display of E on phage particles, the recombinant TG1 were grown in growing medium 2xYT and expression was induced using 1 mM isopropyl-B-D-thiogalactopyranoside (TPTG) for four hours. The TGI culture was then clarified by centrifugation and SDS reducing sample buffer was added to lyse the E. coli pellet and prepare the proteins for SDS- PAGE separation. Proteins that were separated were transferred onto an Immobilon- NC membrane for western blotting. Detection of recombinant E was performed using antibodies specific against the Flag tag engineered at the C-terminus of the recombinant E protein, and using antibodies specific against the phage.
Example 2
The HTS binding and inhibition assay was performed on cell protein lysate. The lysate (1-3 ug) was previously coated in 96-well test plates overnight at 4 °C using 0.05 M sodium carbonate (Na2C03). ph 9.6. Coated cell lysate was blocked with 1% polyvinyl-pyrrolidone (PVP) containing 0.02% tween detergent for two hours at room temperature. Concurrently, circularized peptides CDRL2-SS or CDRH3-SS were added into the recombinant phage mix (~ 5 x 106 particles). They were incubated for two hours at 37 °C. The phage mix was added into the test plates and the test plates were incubated at 37 °C for one hour. Unbound phage were removed using PBS containing 0.05% Tween detergent. Phage that bound to the coated cell lysate was saved by adding TGI E. coli into the test plates and incubating the plate at 37 °C for 30 minutes. The E. coli was then spread on a selective medium agar and the number of colonies was determined the following day. The number of colonies was representative of the number of recombinant phage that bound to the cell protein lysate.

Claims

CLAIMS:
1. A method of identifying a potential reagent for inhibiting interaction between target polypeptides and a recombinant protein comprising the steps of providing a plurality of target polypeptides attached on a solid platform:
bringing a plurality of a potential reagent into contact with and bind onto the recombinant protein;
adding the mix containing the potential reagent and recombinant protein into contact with the target polypeptides;
removing the unbound potential reagent and recombinant protein that is expressed on a phage display system;
adding a plurality of bacterial host cells capable of being infected by the phage to the solid platform; and
determining the presence or number of the infected bacterial cells;
whereby binding of the potential reagent onto the recombinant protein inhibits interaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial host cells, while the incapability of the potential reagent to bind to the recombinant protein allows subsequent infection of the bacterial cells by the recombinant phage.
2. A method according to claim 1, wherein the target polypeptides are directly attached onto a solid platform in the version of microtiter plates.
3. A method according to claim 1 or 2, wherein the target polypeptides are lysate or total proteins of a type of cells containing polypeptides of different conformations.
4. A method according to claim 1 or 2, wherein the phage display system is helper Ml 3 phage.
5. A method according to claim 1 or 2, wherein the bacterial host cells are E.coli TGI .
A method according to claim 1 or 2, wherein the step determining the binding of the recombinant protein to target polypeptides and hence the presence or number of the infected bacterial cells is conducted through the plating of the infected bacterial cells on a suitable selective medium followed by counting of the colonies produced from the plated bacterial cells.
A method of identifying a potential reagent for inhibiting interaction or binding between the target polypeptides and recombinant protein comprising the steps of
providing a plurality of target polypeptides attached on a solid platform;
bringing a plurality of a potential reagent into contact onto the target polypeptides;
removing the unbound potential reagent;
bringing a plurality of recombinant protein into contact with the bound target polypeptides;
removing the unbound recombinant protein;
adding a plurality of bacterial host cells capable of being infected by the phage to the solid platform; and
determining the presence or number of the infected bacterial cells;
whereby binding of the potential reagent onto the target polypeptides inhibits interaction between the target polypeptides and the recombinant protein as well as subsequent infection of the bacterial cells, while incapability of the potential reagent to bind onto the target polypeptides allows subsequent infection of the bacterial cells by the phage display system.
A method according to claim 7, wherein the target polypeptides are directly attached onto a solid platform consisting of microtiter or 96-well plates.
A method according to claim 7 or 8, wherein the target polypeptides are lysate or total proteins of a cell containing polypeptides of different conformations.
10. A method according to claim 7 or 8. wherein the phage display system is Ml 3 helper phage.
11. A method according to claim 7 or 8, wherein the bacterial host cell is E.coli TG1.
12. A method according to claim 7 or 8, wherein the step determining the presence or number of infected bacterial host cells is conducted through the plating of the infected bacterial cells on a suitable selective medium followed by the counting of colonies produced from the plated bacterial cells.
13. A method of identifying a target polypeptide that interacts with the recombinant protein comprising the steps of
providing a plurality of a target polypeptide attached onto a solid platform; bringing a plurality of the recombinant protein expressed on a phage display system into contact with the target polypeptide;
removing the unbound recombinant protein and phage;
adding a plurality of bacterial host cells capable of being infected by the phage to the solid platform; and
determining the presence or number of infected bacterial cells;
whereby capability of the recombinant protein to bind to the target polypeptide allows subsequent infection of the bacterial cells, while incapability of the recombinant protein to bind to the target polypeptide prevents subsequent infection of the bacterial cells by the recombinant phage.
14. A method according to claim 13, wherein the target polypeptide is directly attached onto a solid platform comprising of microtiter or 96-well plates.
15. A method according to claim 13 or 14, wherein the target polypeptide is a homogenous mixture of a purified protein/polypeptide or peptides.
16. A method according to claim 13 or 14, wherein the phage display system is Ml 3 helper phage.
17. A method according to claim 13 or 14, wherein the bacterial host cell is E.coli TG1.
18. A method according to claim 13 or 14, wherein the step deterrrhning the presence or number of the infected host cells is conducted through the plating of the infected bacterial cells on a suitable selective medium followed by .the counting of colonies produced from the plated bacterial cells.
PCT/MY2010/000322 2009-12-15 2010-12-15 A method of high throughput screening of inhibitors that inhibit interaction of an attachment protein to cell receptors using recombinant phage display WO2011074941A1 (en)

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