Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

• This invention relates to methods for screening bacteriophage libraries, to modified bacteriophage libraries for use in such methods, to peptide ligands identified and/or isolated by the described procedures and to the use of identified ligands and consensus sequences thereof in the design of pharmaceutical and diagnostic agents.
• filamentous bacteriophage such as the filamentous coliphage M13 are genetically modified by adding random oiigonucleotides to the coding sequences that encode one of the bacteriophage coat proteins.
• the coat proteins of the bacteriophage particles are consequently expressed with a random collection of N- terminal oligopeptides.
• n The number of possible oiigonucleotides which may be constructed from the 20 naturally occurring amino acids is given by the formula 20 n , where n is the number of amino acids in the oligonucleotide.
• n 7
• a typical library may contain from 10 6 to 10 10 recombinant bacteriophage, each of which is distinguished by a coat protein (e.g. gp3 or gp8 in the case of phage M13) bearing a different N-terminal oligopeptide.
• a coat protein e.g. gp3 or gp8 in the case of phage M13
• every possible oligonucleotide may be represented in the library.
• the number of possible different 7-mers is 20 7 , i.e. 1.28x10 8 combinations and each of the possible sequences may be present in the library.
• Bacteriophage display of peptide libraries is widely used to select peptides that recognise targets.
• the protein is immobilised on a support and the immobilised protein "panned" with a suspension of the library of bacteriophage particles.
• Those bacteriophage having coat proteins with N- terminal sequences which bind to the immobilised receptor protein will be preferentially retained and bacteriophage without recognised N-terminal sequences may be eliminated by washing.
• the conventional approach is to attach the target to a plastic dish and pan the bacteriophage peptide library against this.
• Non- adherent bacteriophage are washed from the plate and bacteriophage that adhere to the target are eluted by washing and the selected bacteriophage are amplified in bacteria.
• the sequence(s) of the recognised N-terminal oligopeptide(s) may then be determined by sequencing the part of the phage DNA that codes for the coat protein.
• Ph.D. ® Phage Display Peptide Library Kit supplied by New England Biolabs. Inc.
• the target protein For conventional panning approaches, the target protein must first be solubilised, isolated and purified.
• the initial solubilisation requires that the plasma membrane is disrupted by the use of detergents.
• Preparation of the target protein for attachment to plastic dishes may destroy the normal architecture and prevent the protein from adopting the correct conformation.
• the native cell expresses many cell surface proteins which will be recognised by peptides and these will be positively selected at the same time as the peptide sequences that recognise the desired target protein.
• the desired cell surface protein may be a minor component on the cell surface, the majority of the selected bacteriophage will be possess peptides that recognise proteins that are not the desired target.
• the "signal" Native cells rarely express a large amount of any one given protein on their cell surface. For instance, a receptor is considered to be abundant if there are 100,000 copies per cell. Other proteins and receptors may be very rare species. For instance, many growth factor receptors are present at low levels on the cell surface. Therefore, the amount of target is very low.
• WO 95/02823 (Brann, M) describes the use of whole cells as substrates for screening peptides, but does not refer to the use of bacteriophage expression libraries.
• WO 92/15702 (Chiron Corp.) describes the screening of bacteriophage libraries against whole cells and discusses the problems associated with the use of whole cells as substrate.
• lines 1 1 to 16 on page 6 state that "[planning against whole cells is problematical because one tends to select compounds having an affinity for the binding moiety present in the highest concentration (e.g., other surface receptors, carbohydrates, and the like).
• moieties may intrinsically be capable of binding a peptide with higher affinity than other moieties (for example, some receptors may have a deep cleft which permits maximal interaction with a peptide)".
• a given peptide which binds specifically to a receptor protein of interest might additionally bind to another receptor protein, for example one of related structure.
• the binding to the other protein of related structure might therefore interfere with the binding of the protein of interest and/or it may be difficult to distinguish between the related proteins.
• a method of producing a modified bacteriophage library suitable for use in combination with cells of a selected strain that have been transformed for expression of heterologous (non native) protein in a screening procedure in which specific binding between an individual bacteriophage of the library and a recognition site of the heterologous protein is detected comprises:
• the invention is especially concerned with elimination of library members which bind to native cell surface associated proteins of the selected strain.
• members of the bacteriophage library which bind to native cell surface associated proteins are bound to the cells, and in step (c) a modified bacteriophage library is isolated which is depleted in library members which bind to native cell surface associated proteins of the selected strain.
• the invention is especially concerned with the use of bacteriophage libraries to distinguish between closely related heterologous proteins, for example between recptors of different sub-types.
• bacteriophage libraries to distinguish between closely related heterologous proteins, for example between recptors of different sub-types.
• more than one receptor sub-types are known, as indicated by the following Table:
• the method of the invention is directed to the production of a modified bacteriophage library suitable for use in combination with cells of a selected strain that have been transformed for expression of a first heterologous (non native) protein (especially a cell surface associated protein) in a screening procedure in which specific binding between an individual bacteriophage of the library and a recognition site of the first heterologous protein is detected, wherein step (a) comprises subjecting an initial library containing said individual bacteriophage and other bacteriophage to a fractionation procedure which comprises contacting the initial bacteriophage library with cells of said selected strain which do not express said first heterologous protein, but which do express a second heterologous (non native) protein, whereby members of the bacteriophage library which bind to said second hetereologous protein are bound to the cells, and in step (c) a modified bacteriophage library is isolated which is depleted in library members which bind to said first heterologous protein.
• the first and second heterologous proteins may be receptor proteins of different subtypes, whereby in step (c) a modified bacteriophage library is isolated which is depleted in library members which bind to heterologous proteins of the subtype comprising said first heterologous protein.
• step (a) the initial library is contacted with cells which have been transformed so as to express the second heterologous protein (which may, for example be a sub-type of a receptor protein).
• Library members which bind specifically to that subtype may then be eliminated from the library and the resulting depleted library used in a subsequent detection step that is aimed at selecting library members which bind to the first heterologous protein.
• the depleted initial library is contacted with cells which have been transformed so as to express the second heterologous protein (which may, for example be a sub-type of a receptor protein).
• Bacteriophage which are selected by this step will be characterised by the ability to bind to the second heterologous protein, but not to the first. As will be appreciated, this technique is of especial use in distinguishing between receptor subtypes.
• the bacteriophage library used in either embodiment would generally comprise a plurality of members possessing a random collection of oligopeptides expressed at the N-terminus of a coat protein.
• the oligomers may, for example be from 7 to 18 amino acids in length, specific examples being 7-mers and 15-mers.
• the initial step (a) consists of fractionation procedure which comprises contacting the initial bacteriophage library with cells of said selected strain which do not express said heterologous protein, whereby members of the bacteriophage library which bind to native cell-surface associated proteins are bound to the cells.
• the cells of the selected strain which do not express the heterologous protein are preferably what we refer to as "mock transfected cells", i.e. cells that have been transfected with vector which is incapable of transforming the cells to express said heterologous cell surface-associated protein.
• the selected strain of cells which are used to express the heterologous protein may be of diverse origins, (e.g. mammalian, avian, insect, yeast, etc.), but conveniently, any established line of cells with a well characterised associated expression system is used. Most preferably, mammalian cells, especially primate cells are used.
• COS cells One particular class of cells that is convenient to use are so-called COS cells, for which a well understood transfection and expression system is available.
• COS cells were originally derived from African Green Monkey kidney and are stably transformed with SV40 virus. The cells express the SV40 large T antigen and are capable of effecting the transient expression of cDNA introduced in the form of an SV40-derived vector in which the cDNA is operatively linked to the SV40 origin of replication.
• Examples of COS cell lines are the lines COS1 and COS7, each of which is obtainable from ATTC. Other cells that may be used are murine cells such as the WOP cell line.
• Vectors suitable for expressing heterologous cDNA in COS cells are described by Seed era/, in Nature (1987), 329:840-842 and Proc. Natl. Acad. Sci. 84:3365-3369 and are available from the authors thereof.
• One example of the vectors available from this source is the so-called CDM vector (also known as 77H3M).
• CDM vector also known as 77H3M
• Another specific vector suitable for transforming COS cells is the vector pCDM8, which is a derivative of CDM, and is available commercially from Invitrogen.
• heterologous protein may represent as much as 2% of the cell surface proteins.
• the DEAE/dextran method of transformation is thus preferably used in carrying out the method of the invention.
• the efficacy of the method of the invention has been demonstarted by using as target, the human high affinity receptor for immunoglobulin G (IgG), expressed in COS cells.
• the step defined in accordance with the invention of subjecting the initial library containing said individual bacteriophage and other bacteriophage to a fractionation procedure which comprises contacting the initial bacteriophage library with cells of said selected strain which do not express said heterologous protein, whereby members of the bacteriophage library which bind to native cell-surface associated proteins are bound to the cells, may be termed a "pre-clear" step, which provides the important technical advantage of reducing "noise".
• the "noise" component in the approach is reduced in accordance with the invention by employing a "pre-clear step”; thus, the bacteriophage library is first incubated with normal cells or mock transfected cells to remove from the bacteriophage library a substantial proportion, and in many instances, all those phage that bind to cell surface components that are naturally expressed, or bacteriophage that express "sticky" peptides.
• knock transfected is meant cells that are transfected with an "empty" vector", i.e. a vector which has not been loaded with cDNA of a heterologous protein.
• the bacteriophage library is first incubated with cells that have been transfected with said second heterologous protein to remove from the bacteriophage library a substantial proportion, and in many instances, all those phage that bind to said second heterologous protein.
• the modified bacteriophage library that is recovered after the "pre-clear" steps referred to is a new product, which itself forms a further independent aspect of the present invention.
• the invention further provides a modified bacteriophage library obtainable by the methods described above.
• the invention further provides according to the first embodiment, a modified bacteriophage library for use in conjunction with cells of a selected strain that have been transformed for expression of a heterologous (non-native) protein (especially a cell surface associated protein) in a screening procedure in which specific binding between an individual bacteriophage of the library and a recognition site of the heterologous protein is detected, characterised in that the library is depleted in members that bind to one or more proteins other than said heterologous protein.
• a heterologous (non-native) protein especially a cell surface associated protein
• the invention provides a modified bacteriophage library for use in conjunction with cells that have been transformed for expression of a first heterologous (non native) protein (especially a cell surface- associated protein) in a screening procedure in which specific binding between an individual bacteriophage of the library and a recognition site of the heterologous protein is detected, characterised in that the library is depleted in members that bind to a second heterologous (non native) protein (especially a cell surface- associated protein).
• a first heterologous (non native) protein especially a cell surface- associated protein
• modified bacteriophage libraries defined above are of commercial value as it can be amplified and used for future pannings.
• a "positive selection" step is provided in accordance with the invention to identify bacteriophage expressing peptides that recognise the target protein.
• bacteriophage from the original library that do not attach to the mock transfected or non-transfected cells (or to the cells transformed so as to express the second heterologous protein referred to in the second embodiment of the invention) are incubated with cells engineered to express the target protein at high copy number.
• a method of screening a bacteriophage library so as to isolate or identify an individual bacteriophage of the library which displays an oligonucleotide which undergoes specific binding to a ligand recognition site of a protein (especially a cell surface- associated protein), which comprises expressing the protein by transecting a cells of a selected strain, contacting the transfected cells with the library, and selecting bacteriophage on the basis of their capacity to bind to the protein expressed by the transected a cells, characterised in that the bacteriophage library is a modified bacteriophage library as defined herein.
• the bacteriophage are preferably selected by carrying out a plurality of rounds of screening, however it is found that the number of rounds can be substantially reduced compared to prior art procedures in which the bacteriophage library has not been subjected to a "pre-clear" step in accordance with the invention.
• the invention further provides a refinement of the screening procedure which allows peptides which bind specifically to ligand binding sites of the protein (especially a cell-surface associated protein) to be identified.
• This refinement is of importance, because on occasions, peptide sequences in a bacteriophage library may bind specifically to a cell-surface associated protein at a location which is distal from, or not functionally associated with a ligand binding site.
• the selection procedure comprises at least one round of screening which comprises subjecting the target protein to a panning step in which the protein is contacted with the bacteriophage in the library, separating unbound bacteriophage from bacteriophage which are bound to the target protein, and subjecting bacteriophage which are bound to the target protein to a displacement step which comprises contacting bacteriophage which are bound to the target protein with the ligand that corresponds to the ligand receptor site, whereby bacteriophage that are bound at the ligand receptor site are displaced, and recovering displaced bacteriophage.
• the method of selecting from a bacteriophage library individual bacteriophage which specifically bind to a ligand receptor site of a target protein which comprises subjecting the target protein to a panning step in which the protein is contacted with the bacteriophage in the library, separating unbound bacteriophage from bacteriophage which are bound to the target protein, and subjecting bacteriophage which are bound to the target protein to a displacement step which comprises contacting bacteriophage which are bound to the target protein with the ligand that corresponds to the ligand receptor site, whereby bacteriophage that are bound at the ligand receptor site are displaced, and recovering displaced bacteriophage forms a further independent aspect of the invention.
• Peptides identified using the procedures described herein form further aspects of the invention, by virtue of their ability to recognise target proteins. They may thus be used commercially as therapeutic or diagnostic reagent or sequence information contained in the thus isolated or identified peptides may be used in the design of other pharmaceutical products. Options include chemical or genetic modification of the peptide or use of the native peptide itself for diagnostics, magic bullets to deliver agents such as drugs, toxins and antibodies to cells.
• the invention provides a further selection procedure, that may be used independently from, or in combination with the step referred to as the "pre-clear" step.
• This further procedure involves selection of bacteriophage that express peptides that interfere with binding of the natural ligand, drugs or other factors such as antibodies that recognise the target protein.
• the invention further provides method of selecting from a bacteriophage library individual bacteriophage which specifically bind to a ligand receptor site of a target protein, which comprises subjecting the target protein to a panning step in which the protein is contacted with the bacteriophage in the library, separating unbound bacteriophage from bacteriophage which are bound to the target protein, and subjecting bacteriophage which are bound to the target protein to a displacement step which comprises contacting bacteriophage which are bound to the target protein with the ligand that corresponds to the ligand receptor site, whereby bacteriophage that are bound at the ligand receptor site are displaced, and recovering displaced bacteriophage.
• previously selected bacteriophage may be selected that now fail to recognise the target when the cells expressing the target are incubated with the other desired component such as the natural ligand, modified ligand, drug or antibody.
• the subsetof bacteriophage that express proteins which bind to a specific region of the target are defined and selected.
• the peptides identified by the methods of the invention are of particular value as, or in the design of commercial products, as they can be used as ligand or drug mimetics.
• Information obtained from the array of peptides selected in this way can be used to define commonality of shared features and used for design of peptides with distinctive features, peptide-like structures and nonpeptide organics.
• the invention further provides the use of a peptide isolated or identified using the methods described herein in the manufacture of a pharmaceutical composition for treating a disease condition involving the cell surface-associated protein or its ligand.
• the invention further provides the use of a peptide isolated or identified using the methods described herein in the design of manufacture of a pharmaceutically active substance for treating a disease condition involving the cell surface-associated protein or its ligand.
• Figure 1 is a schematic diagram illustrating methods according to the invention
• Figures 2 and 3 are bar charts illustrating the results of selecting bacteriophage using the methods of the invention.
• Figures 4 and 5 show data generated using the BIAcore 2000 affinity analysing apparatus.
• An example of an overall procedure in accordance with the invention is shown in the attached Figure 1.
• the example shown is expression of the DNA for Fc RI COS cells.
• other cell lines can be used throughout this process.
• the bacteriophage library is first incubated at 4°C with COS cells that were mock transfected (i.e. cells were processed through the transfection procedure but no DNA or an irrelevant DNA was transfected into the cells).
• the cells and bacteriophage were kept constantly mixed at 4°C in isotonic buffer at physiological pH.
• the resulting modified bacteriophage library is a new product and can be used for all subsequent manipulations. I.e. we have created a bacteriophage display library where peptide sequences that recognise COS cell surface proteins have been removed.
• the supernatant from the "pre-clear” step was then incubated at 4°C with COS cells expressing the cDNA for a cell surface protein suspended in isotonic buffer at physiological pH. The cells were kept constantly mixed.
• Bacteriophage that express peptides which recognise the target protein are allowed to attach to these transfected cells at 4°C (to prevent internalisation of the target) protein), the cells were washed repeatedly with large volumes of cold isotonic buffer at physiological pH to remove the non-adherent bacteriophage. (We found that it was desirable to wash the cells at least five times before no bacteriophage could be recovered from the wash supernatants) . 5. Adherent bacteriophage were then removed by washing the cells in isotonic solution at low pH (less than pH 4). Cells were separated from the supernatant and the supernatant was neutralised.
• the rounds of positive panning may be repeated, if desired.
• the natural ligand human IgGl
• Fc-yRI bacteriophage expressing peptides that recognise the receptor
• Peptides that interact with the cell surface protein at a site close to or comprising the binding pocket for ligand can be selected through a process of reverse panning. Under these circumstances, it is necessary first to have selected the population of bacteriophage that express peptides recognising the cell surface protein by the positive selection step described above. These bacteriophage are then incubated with COS cells expressing the cell surface protein in the presence of the ligand at 4°C in isotonic buffer. The concentration and nature of the displacing molecule can be altered as necessary to select preferentially different affinity displayed peptides or peptides with different specificities.
• the supernatant and COS cells are separated but the bacteriophage in the supernatant are amplified as these represent bacteriophage that express target specific peptides unable to bind the cell surface protein in the presence of ligand.
• the peptide sequence is derived using standard sequence reactions of the bacteriophage DNA
• a especially valuable procedure that can be used in conjunction with the method of the invention in order to provide additional information about the binding capabilities of oligopeptide sequences identified in the initial screening involves the use of the BIAcore 2000 affinity analysing apparatus. Subsequent evaluation using this apparatus allows a rapid assessment of selected bacteriophage expressing peptides of interest.
• the bacteriophage are captured on to the chip surface using an anti-phage antibody.
• the cell surface-associated protein in solubilised form in the following example Fc-yRI
• Fc-yRI solubilised form
• Example 2 and 3 illustrates the application of the method of the invention to isolate peptide consensus sequences that bind to the FcpRl recognition site.
• a bacteriophage library expressing 15-mer peptides was used for affinity selection with COS cells actively expressing Fc RI-MANX on the cell surface.
• Fc RI -MANX is described in Davis et al., EMBO Journal, 14 (1995) pp.432-441.
• COS cells were split to be approximately 50% confluent the next day for transfection with FcjvRI-MANX construct. 6 sterile 60mm culture dishes.
• COS cells were transected with the Fc RI-MANX construct in the presence of DEAE-DEXTRAN .
• the "pre-clear" step was effected as follows:
• MTC P1 (pellet) dil. range 10 "5 , 10 "6 , 10 “7 , 10 “8 , 10 9 '
• COS cells expressing FCKRI-MANX constuct were lifted off as described in section (i) above, resuspended in 1 ml of cold PBS-BSH and mixed with 2mls of the supernatant from step (v).
• the mixed suspension [COS/FcpRI-MANX and phage] were incubated at 4°C for 1 hr with gentle rotation.
• Cos cells 60mm were split into 6 x 60mm plates to about 50% confluent, next day for transfections.
• FCKRI-MANX DNA was prepared for transfection, 5//I of DNA in 200 /I of TE (one Ependorf for each transfection; 3 were set up). The DNA was diluted in TE as above and 200//I of DEAE-DEXTRAN was added. The solution was left to stand at room temperature for 30 minutes. In the mean time, media aspirated off COS cells and replaced with 1 .5ml of NU medium plus the addition of 1.5 ⁇ l of chloroquine. The cells were left at 37°C for 1 minute. Media was removed from the plates and replaced with 2mls of warm NU media (37°C). The DNA mix was then added drop by drop and the cells incubated at 37 °C for up to 4h.
• the peptide library was amplified. 1 ml of the library was added to 20ml of ER2537 culture at mid-log point and grown at 37 °C with vigorous shaking for 48 hours. The culture was transferred to a Falcon tube and spun at 3,000rpm in a Beckman GS-6KK centrifuge (4°C) for 15 minutes to pellet bacterial cells. The supernatant was transferred to a fresh tube and respun. 80% of the supernatant was transferred to a fresh tube and precipitated phage allowed to form overnight at 4°C (after addition of 1 /6vol. PEG/NaCI).
• the phage-library was cleaned up and titred.
• the PEG precipitated phage were pelleted for 15 minutes at 10,000rpm at 4°C.
• the supernatant was decanted, respun once and the residual supernatant removed with a pipette.
• the pellet (phage) was suspended in 1 ml of sterile PBS. The suspension was transferred to a centrifuge tube and spun at 13,000rpm - Beckman Cold Room, 5 minutes to pellet residual cells.
• the supernatant was then transferred to a fresh 1 .5ml Ependorf tube and precipitated with 1/6th vol PEG/NaCI and incubated on ice for one hour.
• the precipitate was pelleted at 4°C, 13,000 Beckman centrifuge.
• the supernatant was discarded and the pellet suspended in 1 ml of PBS pH 7.4.
• the amplified eluate was titred for second round screen.
• a single colony from a minimum medium plate for strain ER2537 was picked and used to inoclate 5ml of LB.
• the E.coli were allowed to grow for 3 hours until they reached mid-log phase (OD 600-0.5-1.0. Phage were diluted N-10 12 .
• the plates were incubated overnight at 37°C (inverted) in ovens without a fan.
• COS cells were trypsinized and plated onto fresh culture dishes, supplemented with fresh DMEM and incubated at 37 °C.
• MTC Mock transfected COS cells
• PBS PBS/2mM EDTA (2 ⁇ l) per 60mm culture dish. This was enhanced by incubation for 10 minutes at 37°C. Removal of cells from the surface was checked by microscope. The cells were pelleted 1 ,000rpm x 5 min. at 4°C. The cells were resuspended in PBS:BSA (1 %), (10ml) and washed 1 x by gentle inversion on ice. The cells were pelleted by centrifugation as before. The resultant pellet was resuspended gently in 500//I of PBS/BSH cold (4°C).
• the MTC-S1 (1 .49mls) was obtained.
• Transfected cells with FCKRI-MANX (CFM) were removed from the 60mm dishes (x3 as described above for MTC cells).
• the CFM cells were suspended in 500 ⁇ l of PBS-BSA 4°C and MTC-S1 (1.490mls) was added. The resultant mix was gently rotated for 1 h at 4°C.
• the CFM/Phage mix was centrifuged at 1 ,000rpm for 5 minutes at 4°C.
• the CFM-S1 (supernatant) was carefully removed and retained for titring.
• the CFM pellet/phage was washed 5x (W1 -W5) with 10mls of PBS-BSA (4°C). The washings were performed on ice, by gentle inversion spinning down between washes.
• the pellet CFM after the final wash was suspended in 1 ml of acidified PBS pH2.5 fora 5 min at 4°C. The solution was mixed gently by inversion. The CFM/PBS 2.5 was centrifuged at 1 ,000rpm x 5 minutes at 4°C to pellet the COS cells. The CFM-P1 and CFM-E1 were retained for titring. The eluate « 1 ml was neutralised with 250//I of 1 MTris-HCI pH 9.2.
• COS cells (8 x 60mm plates) were split into 6 x 100ml plates to be approx 50% confluent by next day. Each 17-mer and 15-mer peptide library was amplified.
• FCKRI-MANX DNA was prepared for transfections 5 ⁇ l of DNA in 200 /I TE (1 Ependorf per each transfection (4 set up)). The DNA was eluted in TE as above and 200 ⁇ of DEAE Dextran was added. The solution was left to stand at room temperature for 20 minutes . In the meantime, media was aspirated off the COS cells and replaced with 1 .5 ml of NU medium plus the addition of 1 .5 ml of chloroquine. Included were COS cells mock- transfected with the above excluding DNA. The cells were left at 37°C for 1 minute . Media was removed from the plates and replaced with 2ml of warm NU medium (37 °C). The DNA mix was then added drop by drop and the cells incubated at 37 °C for up to 4 hours.
• DMSO/PBS shocked - NU medium was removed and replaced with 10% DMSO/PBS solution, and incubated for 2 minutes.
• the phage libraries were cleaned up (filtered 4.5 ⁇ m filter) see p 40.
• COS cells were trypsinised and plated with fresh culture medium supplemented with fresh DMEM and incubated at 37°C . Amplified phage from the day before were titred.
• the phage/MTC suspension was centrifuged at 1000 rpm at 4°C for 15 minutes . Fractions from the MTC-P1 and MTC-S1 were saved for titring.
• COS cells transfected with CKRI-MANX DNA were incubated near the end lgG1 (human) 500 ⁇ l of CFM cells with PBS- BSA - 5:100 dilution of human IgGl (3.3x10-7) saturation, 4°C all incubations were carried out in the cold room on ice at 4°C.
• the MTC-s1 fraction for each library ( ⁇ 1.5ml) was added to the 500 ⁇ l of COS cells (CFM) preincubated with lgG1 and rotated gently at 4°C for 1 hr.
• the COS cells were pelleted by centrifugation as before and the supernatant retained for titering. The phage blocked in their binding to FCKRI. To test for a lack of internalisation, The COS cells in the pelleted fraction were goat anti-human Igg1 FITC-labelled and checked by microscopy-. Result produced the ring structure around cells (labelling normally produced suggesting no receptor internalisation)
• PBS Phospate buffered saline (8g of NaCl, 0.2g of KCI, 1.44g of Na 2 HPO4,
• bacteriophage having peptides with the following sequences were identified (multiple sequence alignment of individual 15-mer peptide sequences are shown):
• Consensus P A P L L P Following further analysis of the recovered bacteriophage using the BIAcore 2000 affinity analysing apparatus (see Figures 4 and 5 for data generated), phage K1, K5, K25 abd K26 were selected as having specific relatedness to the lgG1 binding site of the FCKRI protein.
• the ALRHPPP peptide qequence was first observed in round 4 of panning and became increasingly predominant in subsequent rounds of panning, to be the only sequence found in the lasr round, cycle 7.
• the TMSDPHS peptide sequence was observed with increasing frequency in rounds 4, 5 and 6 of biopanning

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Biomedical Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Virology (AREA)
• Crystallography & Structural Chemistry (AREA)
• Toxicology (AREA)
• Analytical Chemistry (AREA)
• Plant Pathology (AREA)
• Immunology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=10816703

Family Applications (1)

Country Status (4)

Cited By (1)

Families Citing this family (2)

Family Cites Families (1)

• 1997
  • 1997-07-30 GB GBGB9716094.9A patent/GB9716094D0/en active Pending
• 1998
  • 1998-07-29 EP EP98936536A patent/EP1000142A1/de not_active Withdrawn
  • 1998-07-29 AU AU85503/98A patent/AU8550398A/en not_active Abandoned
  • 1998-07-29 WO PCT/GB1998/002269 patent/WO1999006542A1/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events