Patent Application
20100273669
The present invention relates to phage display.
DNA recombination and genetic engineering techniques make it possible today to modify the structure of recombinant proteins or antibodies and evolve their functions. This is made possible by the contribution of modifications on the DNA sequence of a gene encoding for the aforementioned protein. These modifications corresponding to the creation of mutations can be carried out in a site directed or completely random way (for review see 1-3) and generate mutant libraries. The screening of these libraries allows selection of mutants presenting the required function.
One of the recent applications is the generation of recombinant antibody libraries. Libraries are generated starting from mRNA extracted from B cells, (from diverse lymphoid sources, taken among healthy subjects or patients suffering from various diseases) by PCR-based or similar cloning technology (4-6). These libraries can be optimised in term of diversity by the random incorporation of mutations on the heavy chains (VH) and light chains (VL) variable domains of immunoglobulins. Antibody libraries can be expressed as variable fragments (VH, VL, scFv or Fab). VH and VL variable domains of the antibody are responsible for the recognition and the binding to the antigen. Genetic engineering of this region helps the optimization of the immunologicals properties such as affinity, stability and specificity of an antibody for an antigen (7,8). The same approach is considered for the constant region (Fc region) of an antibody which carries binding epitope for many receptors, like effector cells of the immune system (for review see 9 and references therein).
Recombinant antibody libraries, naive or optimised by random mutagenesis, are of very significant size and very powerful selection tools are required in order to isolate the antibody of interest. Many of the selection platforms used today (bacterial, yeast and phage display) share four key steps: generation of genotypic diversity, coupling genotype to phenotype, application of selective pressure and amplification. Systems used today work on the basis of antibody expression (VH, VL, Fab or scFv fragment) on the surface of a cellular (bacterium, yeasts) or viral (phage) system. Phage display is the most popular system for antibody library screening (10) and relies on a strong binding of the antibody to the antigen which also makes it well suited to affinity maturation. However, this requires that the interaction between the antigen and the antibody is strong enough to be maintained until the end of the screening process and to allow the selection of the required antibody expressed on the phage cell-surface. In addition, when the antigen is a protein, the screening/selection process from an antibody library involves non specific or a specific interactions which can generate many false positives. Thus, difficulty lies in the selection of mutants presenting a specific interaction with the antigen (or protein). In most of the current selection systems, identification of the specific interaction among the large non specific interactions requires many long and tedious stages.
In order to overcome these disadvantages, EP0614989 and some publications (11-13) describe a method for the selection of proteins which are involved in protein-ligand interaction. This method relates to the recovery of the infectious character of a phage displaying on its surface recombinant antibody fragment. The interaction between the antibody displayed on the phage surface and its ligand allows the restoration of the phage infecting ability. Indeed, this interaction occurs with the bringing together of two fragments of a viral coat protein (e.g. the minor coat protein pIII) which is essential to the phage infecting ability. However, this approach also suffers from several disadvantages. First, the infecting ability of the phage depends on strength of the interaction between the displayed protein and the ligand. Consequently, only strong or very strong affinity interactions will be able to keep together the two viral coat infectious protein fragments and restore the infecting ability of the phage. The outcome is a significant loss of interesting mutants in term of specificity. Mutants having a moderate to strong affinity, but being able to be the subject of an improvement during an additional mutagenesis-selection cycle will not be selected. In the case of a naive or randomly evolved antibody library, selection of an antibody with strong affinity to the antigen generally requires generation of different large size libraries and several mutagenesis-screening cycles to increase the success rate.
Furthermore, in the reaction medium containing the mutant library an important part of the ligand fused to the fragment of the viral coat protein remains free. During the selection step, this fusion molecule can bind to the host cells likely to be infected by the phages. Hence a competition with the phages with restored infecting ability takes place. There is then a phenomenon of exhaustion of the possibilities of connection to the host cell for the infectious phages. Thus, one observes a loss of a considerable proportion of the mutants with specific binding to the ligand.
Consequently it remains tedious to identify a peptide or a polypeptide which binds to a target from a random mutant library even using the more up to date phage display published methods. It is the object of the present invention to devise an improved method for selecting from a random protein variant library a peptide or polypeptide which binds to a target of interest.
The present invention provides a versatile and sensitive method for selecting a peptide or polypeptide which binds to a target. The invention is based on protein trans-splicing and phage display.
Protein splicing is defined as the excision of an intervening sequence (the INTEIN) from a protein precursor and the concomitant ligation of the flanking protein fragments (the EXTEINS) to form a mature protein (extein) and the free intein. The intein plus the first C-extein residue (called the +1 amino acid) contain sufficient information to mediate splicing of the intein out of the protein precursor and ligation of the exteins to form a mature protein. Intein-mediated protein splicing results in a native peptide bond between the ligated exteins. It is now known that inteins incorporated into non-native precursors can also cause protein-splicing and excision of the inteins. In addition, an N-terminal intein fragment in a fusion protein and a C-terminal intein fragment in another fusion protein, when brought into contact with each other, can bring about trans-splicing between the two fusion proteins.
Thus, in accordance with the present invention, the protein splicing feature is used in vitro to transform a non-infectious virus into an infectious virus, thereby allowing the selection of a positive interaction of a peptide or polypeptide with a target. By using this method, extremely large libraries can be screened.
The present invention ensures a positive selection of the peptides or polypeptides of interest. The present invention allows the selection of peptides or polypeptides with a good specificity for a target and permits the improvement of their affinity for the target by successive mutagenesis rounds. The present invention is therefore well-suited to affinity maturation of antibodies in multiple rounds of mutation and selection.
The present invention provides a kit for selecting a peptide or polypeptide which binds to a target. The kit comprises:
a library of viruses each displaying on its surface a chimeric polypeptide of formula X-I1-Z wherein X is a peptide or a polypeptide, I1 is a first fragment of an intein and Z is a peptide or a protein which is present at the surface of each of said viruses, wherein each said virus comprises a nucleotide sequence encoding X and is not able to infect a host cell; and
an adapter molecule of formula A-I2-C wherein A is a molecule which, when A is displayed on the surface of each said virus, renders the virus able to infect said host cell, I2 is a second fragment of said intein and C is a target molecule, wherein X-I1-Z and A-I2-C are constructed in such a way that if X binds to C, A is covalently linked to Z upon trans-splicing through the first and the second fragments of said intein.
Typically the kit further comprises said host cell. Said host cell can be for example a prokaryote host cell and more particularly a bacterial host cell.
Typically any type of target molecule can be used. C can, for example, be selected from the group consisting of an antigen, an antibody, a nucleotide sequence, a receptor.
The three different components X, I1, Z of the chimeric polypeptide of formula X-I1-Z can be directly linked or linked via a spacer comprised of a peptide of 1 to 20 amino acids.
The three different components A, T2, C of the adapter molecule can be directly linked or linked via a spacer comprised of a peptide of 1 to 20 amino acids. Alternatively the components can be linked together by using an appropriate chemical linking agent.
The virus to be used in the present invention can be any virus or viral vector. In a preferred embodiment the virus is a filamentous bacteriophage. For example said filamentous bacteriophage can be selected from the group consisting of Ff filamentous phage, lambda and T7. In particular, said filamentous bacteriophage is a Ff filamentous bacteriophage selected from the group consisting of fd, M13 and fl.
It falls within the ability of the skilled person to select Z, which is a protein or a peptide present at the surface of the virus. Z can be, depending on the virus used, a viral coat protein, a protein of the envelope of the virus, a protein of the capsid or a fragment thereof.
It falls within the ability of the skilled person to select molecule A which, when displayed on the surface of a virus renders the virus able to infect a host cell Techniques and molecules for altering the tropism of a virus are well known (see for example EP1191105 and WO2005040333). Molecule A, for example, can be selected from the group consisting of an antibody, a viral coat protein, a protein of the envelope of the virus, a protein of the capsid and fragment thereof.
In a preferred embodiment, Z is the C-terminal part of a surface protein of a virus which is required by said virus for the infection of a host cell and A is the N-terminal part of said surface protein.
For example if said virus is a filamentous bacteriophage, said surface protein can be selected from the group consisting of protein III (pIII) or protein VIII (pVIII). pIII of bacteriophage M13 comprises three domains of 68 (N1), 131 (N2) and 150 (CT) amino acids. pIII can be easily engineered in two pieces A and Z: the N-terminal part comprising domains N1 and N2: A and the C-terminal part comprising domain CT: Z. A phage only expressing at its surface the C-terminal part of pIII can not infect its traditional host cell. Infecting ability is restored when the N-terminal part of pIII is linked the C-terminal part of pIII.
In a preferred embodiment, X is an immunoglobulin, or a member of the immunoglobulin super-family, or any fragment thereof. In this context, the term immunoglobulin includes members of the classes IgA, IgD, IgE, IgG, and IgM. The term immunoglobulin super-family refers to all proteins which share structural characteristics with the immunoglobulins, including, for example, the T-cell receptor, or any of the molecules CD2, CD4, CD8 etc. Also included are fragments which can be generated from these molecules, such as Fv (a complex of the two variable regions of the molecule), single chain Fv (an Fv complex in which the component chains are joined by a linker molecule), Fab, F(ab′)2 or an immunoglobulin domain, such as the constant fragment (Fc), the variable heavy chain domain (VH) or the variable light chain domain VL.
It falls within the ability of the skilled person to select an intein and the two fragments thereof in order to construct X-I1-Z and A-I2-C in such a way that if X binds to C, A is covalently linked to Z upon trans-splicing through the first and the second fragments of said intein.
Typically the skilled person will use existing protocols to select the two fragments 1 and I2 and construct X-I1-Z and A-I2-C. Protein trans-splicing is a well known technique which has found a variety of applications including in vitro protein semisynthesis (21), segmental isotopic labeling (22), two and three hybrid strategies for monitoring protein activity in vivo (20, 23) and protein cyclization (24). Protein splicing permits the translation of an interaction event into a detectable signal through the reconstitution of a functional protein such as EGFP in E coli and yeast, and firefly luciferase in mammalian cells (see 23, 25-27 and EP1229330). In protein trans-splicing, a protein is split into two fragments and each half is fused to either the N-terminal or C-terminal fragments of an intein. Some inteins like the cis-splicing VMA intein from Saccharomyces cerevisiae have been engineered to be split in two fragments (N- and C-intein) to produce in vivo trans-spliced recombinant proteins (20). N-intein or C-intein alone is incapable of catalyzing protein splicing. However, when the N-intein and a C-intein, fused respectively to two interacting proteins, are in close proximity, they are capable of catalyzing protein trans-splicing.
Since the initial discovery of the VMA1 intein (14, 15), inteins have been identified in bacteria, archea and eukaryotic unicellular organisms (see The Intein Database and Registry http://www.neb.com/neb/inteins.html). Three Regions are found in each Intein: an N-terminal Splicing Region, a central Homing Endonuclease Region or a small central Linker Region, a C-terminal Splicing Region. Remarkably, inteins as small as 134 amino acids can splice out of precursor proteins. The discovery of mini-inteins and mutational analysis have indicated that the residues responsible for protein splicing are present in the N-terminal Splicing Region and the C-terminal Splicing Region (including the +1 amino acid in the C-extein). Several conserved motifs have been observed by comparing intein amino acid sequences. A nomenclature for these motifs has been defined (16): Blocks A, B, C, D, E, H, F, G. The N-terminal Splicing Region is about 100 amino acids and begins at the intein N-terminus and ends shortly after Block B. The intein C-terminal Splicing Region is usually less than 50 amino acids and includes Blocks F and G. The N-terminal Splicing Region and the C-terminal Splicing Region form a single structural domain, which is conserved in all inteins studied to date.
Mini-inteins are usually about 130-200 amino acids. However, most inteins are greater than 300 amino acids, while the Pab RFC-2 intein is 608 amino acids. These big inteins have a larger linker region between intein Blocks B and F that includes intein Blocks C, D, E, and H homing endonuclease motifs.
The consensus sequence for blocks A, B, F and G is indicated below. Although no single residue is invariant, the Ser and Cys in Block A, the His in Block B, the His, Asn and Ser/Cys/Thr in Block G are the most conserved residues in the splicing motifs. Any member of an amino acid group may be present in the remaining positions, even when a specific predominant residue is indicated.
The upper case letters represent the standard single letter amino acid code for the most common amino acid at this position and lower case letters represent amino acid groups: x: any residue; x1: C, S or T; h: hydrophobic residues: G,A,V,L,I,M; p: polar residues: S,C,T; a: acidic residues: D or E; r: aromatic residues: F,Y,W)
Block A (SEQ ID NO:1): x1hxxDpxhhhxxG (the first residue corresponds to the intein N-terminus)
Block B (SEQ ID NO:2): GxxhxhTxxHxhhh (usually 70-105 residues from N-terminus)
Block F (SEQ ID NO:3): rVYDLpV[1-3 residues]axx[H or E]NFh
Block G (SEQ ID NO:4): NGhhhHNp (p belongs to the downstream extein N-terminus)
In a preferred embodiment the intein is selected from the group consisting of DnaE, Ctr VMA, Mtu recA and Tao VMA.
In a preferred embodiment, Z is linked to the C-terminus of I1 and I1 comprises block F and block G and molecule A is linked to the N-terminus of I2 and I2 comprises block A and block B. Alternatively Z is linked to the N-terminus of I1 and I1 comprises block A and block B and molecule A is linked to the C-terminus of I2 and I2 comprises block F and block G.
In a further embodiment, the present invention relates to the virus comprising a nucleotide sequence encoding X and displaying on its surface a chimeric polypeptide of formula X-I1-Z as defined in the above mentioned kit.
In a further embodiment, the present invention relates to a library of viruses comprising a nucleotide sequence encoding X and displaying on its surface a chimeric polypeptide of formula X-I1-Z as defined in the above mentioned kit.
In a further embodiment, the present invention relates to the adapter molecule of formula A-I2-C as defined in the above mentioned kit.
In a further embodiment, the present invention relates to a vector comprising a nucleotide sequence encoding I1-Z, wherein the vector is capable of being packaged into a virus and wherein the vector comprises a cloning site which enables the introduction of a nucleotide sequence encoding a peptide or polypeptide X in such a way that the chimeric polypeptide X-I1-Z is displayed at the surface of said virus when said vector is packaged.
Typically the vector is a phagemid.
In a further embodiment, the present invention relates to a vector comprising a nucleotide sequence encoding X-I1-Z, wherein the vector is capable of being packaged into a virus and wherein X-I1-Z is displayed at the surface of said virus when said vector is packaged.
In a further embodiment, the present invention relates to a library of vectors comprising a nucleotide sequence encoding X-I1-Z, wherein the vector is capable of being packaged into a virus and wherein X-I1-Z is displayed at the surface of said virus when said vector is packaged.
In a further embodiment, the present invention relates to an expression vector comprising a nucleotide sequence encoding A-I2, wherein said expression vector comprises a cloning site which enables the introduction of a nucleotide sequence encoding a target peptide or polypeptide C in such a way that a chimeric polypeptide of formula A-I2-C can be expressed in a host cell. Typically this expression vector can be used for the production of the adapter molecule.
In a further embodiment, the present invention relates to a kit comprising:
In a further embodiment, the present invention relates to a method for producing a virus as defined above comprising the step of genetically modifying a virus in such a way that when the virus is assembled the chimeric polypeptide of formula X-I1-Z is displayed on the surface of the virus. Typically the step of genetically modifying the virus can be performed by using the vector defined above.
In a further embodiment, the present invention relates to a method for producing a library of viruses as defined above comprising the steps of:
a) generating a library of vectors as defined above, wherein each vector of the library comprises a variant nucleotide sequence encoding X;
b) genetically modifying viruses in such a way that when the viruses are assembled a chimeric polypeptide of formula X-I1-Z is displayed on the surface of the viruses.
Typically the libraries result from the construction of nucleotide sequences repertories, nucleotide sequences characterised in that they are different by at least one change. The generation of the variant nucleotide sequences encoding X may be performed by site-directed mutagenesis, preferentially by random mutagenesis. Random mutagenesis can be performed by using a mutase, Po1 beta for example (see WO0238756).
In a further embodiment, the present invention relates to a method for selecting a peptide or polypeptide X which binds to a target C or a nucleotide sequence encoding X comprising the steps of:
a) combining the different components of the kit defined above comprising a library of viruses and an adapter molecule, where said adapter molecule selectively interacts with viruses displaying a peptide or polypeptide X which binds to C, thereby conferring to these viruses the ability to infect the host cells;
b) replicating the viruses which are infective for the host cells by culturing the viruses in the presence of said host cells;
c) isolating from said host cells the viruses which replicate;
d) determining the nucleotide sequence encoding X from the viruses isolated in step c).
Optionally after step a) and before step b) the adapter molecules not having interacted with the viruses are removed.
In a further embodiment, the present invention relates to a method for producing a peptide or polypeptide X which binds to a target C comprising the steps of:
a) selecting the peptide or polypeptide X by performing the method described above; and
b) producing X.
In the following, the invention will be illustrated by means of the following non-limiting examples as well as the non-limiting figures.
In the following description, all molecular biology experiments are performed according to standard protocol (28).
The intein used was a yeast VMA1-derived intein (VDE or PI-SceI) cloned in a pGEX vector: pGEX-VDE.
The protein III (abbreviated as pIII, gIIIp or g3p) of bacteriophage M13 consists of three domains of 68 (N1), 131 (N2) and 150 (CT) amino acids, connected by glycine-rich linker of 18 (G1) and 39 (G2) amino acids.
a. Insertion of the Intein in the Gene III Protein.
The gene of protein III (gene III) of bacteriophage M13 was PCR amplified and cloned in a pSK vector: pSK-GIII. Site directed mutagenesis was used to introduce SphI and AgeI restriction sites in the glycine-rich linker G2 of gene III using the primer pair: 5′-GGCGGTTCTGAGGGTGGCGCATGCGAGGGAGGCGGCGGTTCCGG-3′ (SEQ ID NO:5) and 5′-COGGAACCGCCTCCOTCGCATGCGCCCACCOTCAGAACCGCC-3′ (SEQ ID NO:6) and the primer pair 5′-GAGGGAGGCGGTACCGGTGGTGGCTCTGG-3′ (SEQ ID NO:7) and 5″-CCAGAGCCACCACCGGTACCGCCTCCCTC-3′ (SEQ ID NO:8), respectively.
The N-terminal domain of the VDE (N-VDE: amino acids 1 to 187) were PCR amplified from pGEX-VDE using the primer pair: 5′-GCATGCTTTGCCAAGGGTACCAATG-3′ (SEQ ID NO:9) and 5′-CTCGAGTGTGCCGTTGCCGTTGTTTCTGTCATTCTCATAAAGAATTGGAGCG-3′(SEQ ID NO:10) which allows to add SphI and XhoI restriction sites at the two extremities of the N-VDE.
The C-terminal domain (C-VDE: amino acids 388 to 455) of the VDE was amplified using the primer pair CTCGAGAGAAACAACGGCAACGGGAACGGCACAGGAGATGTTTTGCTTAACGT (SEQ ID NO:11) and ACCGGTACCGCCTCCCTCGCAATTGTGGACGACAACCTGGGATCC (SEQ ID NO:12) which allows to add XhoI and AgeI restriction sites at the two extremities of the C-VDE and a linker at the N-terminal part of the C-VDE.
The N-terminal and the C-terminal domains of the VDE amplified from the plasmid pGEX-NVDE were then cloned into the SphI-AgeI restriction sites of the gene III to obtain the vector pNTg3p-VDE-CTg3p (
b. Construction of the Phagemid with C-Extein of the VDE in Fusion with CT of pIII.
The C-VDE and CT of pIII (CTg3p) fusion protein was PCR amplified from the vector pNg3p-VDE-CTg3p using the primer pair 5′-ATAAGAATGCGGCCGCATAGAGAAACAACGGCAACGGGAACGG-3′ (SEQ ID NO:13) and TAATACGACTCACTATAGGG (SEQ ID NO:14), which allows to replace XhoI by NotI at the N-terminal and cloned between the NotI and ClaI restriction sites of the vector pSK-GIII in fusion with a signal sequence pelB and under the control of a Lac promoter to generate the phagemid pCVDE-CTg3p (
c. Construction of the Vector to Express the Target in Fusion with N1-N2 Domain (NTg3p) of gIIIp the Half VDE (N-VDE).
The N1-N2 domain of the gene III was PCR amplified from the vector pNP3-VDE-CP3 using the primer pair 5′-CCATGGCTGAAACTGTTGAAAGTTGTTTAGC-3′ (SEQ ID NO:15) and 5′-CTCGAGGCATGCGCCACCCTCAGAACC-3′ (SEQ ID NO:15) which allows to add NcoI in 5′ and XhoI in 3′ and cloned in the NcoI and XhoI restriction sites of the pGEX vector to generate the control plasmid pGEX-NTg3p (
The N1-N2-NVDE fusion protein gene was PCR amplified from the vector pNTg3p-VDE-CTg3p using the primer pair 5′-CCATGGCTGAAACTGTTGAAAGTTGTTTAGC-3′ (SEQ ID NO:17) and 5′-CTCGAGTGTGCCGTTGCCGTTGTTTCTGTCATTCTCATAAAGAATTGGAGCG-3′ (SEQ ID NO:18) in order to insert NcoI in 5′ and cloned in the NcoI and XhoI restriction sites of the pGEX vector providing the plasmid pGEX-NTg3p-NVDE (
The N1-N2-NVDE fusion protein gene was PCR amplified from the vector pGEX-NTg3p-NUDE using the primer pair 5′-TATAGTATGAGCTCGCCATGGCTGAAACTGTTGAAAGTTG-3′ (SEQ ID NO:19) and 5′-TATATAGAATTCTCACTTCTTCTCGAGTGTGCCGTTCCCGTT-3′ (SEQ ID NO:20) in order to insert Seal in 5′ end and EcoRI, and 2 codons for lysine in 3′ end and cloned in the Seal and EcoRI restriction sites of the expression pMG20 vector (MilleGen) providing the plasmid pMG20-NTg3p-NVDE_K.
a. Phage Fusion Antibody Anti-NVEGF
The retrotranscript of the VH and VL genes of the hybridoma VEBA76.50 were PCR amplified and a single chain antibody Fv fragment (scFv) having the structure VH-VL was cloned into the vector pCR4-topoTA (Invitrogen). The VEBA76.50 scFV was digested with NcoI and NotI and cloned into the phagemid pCextein-CTg3p digested with the same enzymes giving the phagemid pCVDE-CTg3p-VEBA76.50 (
This phagemid encodes the VEBA76.50 scFv as an N-terminal fusion of C-terminal domain of the intein (CVDE) and the C-terminal domain of the pIII. Phage particles displaying the scFv on their surfaces were produced in the E. coli XL1blue harbouring the plasmid pCVDE-CTg3p-VEBA76.50 and co-infected with the hyperphage M13KO7ΔpIII (Progen). The phages were then prepared according to standard methods (28).
Competitive ELISA was used to characterise the phage particles displaying on their surface the fusion protein scFv-CVDE-CTg3p. The phage particles were added to each well of microtiter plates previously coated with the fusion protein GST-NVEGF and incubated 2 h at 37° C. in the presence of decreasing concentration of soluble GST-NVEGF used as competitor. After three washes, phages that bound to the wells were detected with a peroxidase conjugate anti-M13 antibody and TMB (Sigma). The inhibition curves obtained permit to determinate the relative affinity of the VEBA76.50-CVDE-CTg3p-Phages for the N terminal part of the VEGF (NVEGF). In this case the deduced relative affinity for the NVEGF was in the nanomolar range (10 nM).
b. Construction of the Target Complex
The N portion of the VEGF was PCR amplified from the vector pGEXNVEGF using the primer pair 5′-CTCGAGCGGCGGCGGACAGTGGACGCG-3′ (SEQ ID NO:21) and 5′-GCGGCCGCTTACCGGGCCAGGGCCTGGGGAGC-3′ (SEQ ID NO:22) was cloned into the vector pCR4-Topo.
The NVEGF was digested with XhoI and NotI and cloned into the pGEX-NTg3p-NVDE digested with the same enzymes, giving the vector pNTg3p-NVDE-NVEGF (
The target fusion complex NTg3p-NVDE-NVEGF produced in E coli strain BL21(DE3) are purified using a glutatione chromatography according to standard methods (28).
A competitive ELISA was done to evaluate the binding of the phage particles to the target fusion complex. The protocole was the same as described previously but in this case the wells were coated with the target complex fusion (GST-NTg3p-NVDE-NVEGF). As a result, the phage displaying the antibody fusion complex binds specifically the N terminal part of the VEGF of the target fusion complex.
c. Formation of a Covalent Link Via Trans-Splicing Following Protein-Target Interaction
During trans-splicing process, the intein was reconstituted and a covalent link occurred with the flanking sequence named extein. The formation of a covalent link between two protein parts can be demonstrated by a particular type of ImmunoAssay (
a. Phage Fusion Complex
A peptide anti-RhoB was isolated from a highly diversify antibody library (MutalBank-Millegen) through a screening against RhoB protein. The peptide R3 (25 aa) has a specific affinity against the protein RhoB. The peptide R3 was PCR amplified with the primers pair 5′-GCAGCCCCATAAACACACAGTATGT-3′ (SEQ ID NO:23) and 5′-ATATATATGCGGCCGCCTTATCGTCATCGTCGTACAGATCTGAACCGCCTCCACCACTC CGCTCGAGGAGATGGATTGTAGCGCTTATCATC-3′ (SEQ ID NO:24) in order to insert NotII, a GS linker, a TAG (Xpress) in 3′ and cloned in the BglII and NotI restriction site of the phagemid pCextein-CTg3p-hinge-Fc to obtain pCVDE-CTg3p-R3 (
b. Construction of the Target Complex
RhoB gene was PCR amplified from the vector pIRES-puro-HA-RhoB (29) using the primer pair 5′-TATAGGTCGACATGGCTTACCCATACGATGTTCCAGA-3′ (SEQ ID NO:25) and 5′-TATATATCTAGATAGCACCTTGCAGCAGTTGATGCA-3′ (SEQ ID NO:26) and was cloned into the vector pCR4-topoTA (Invitrogen). The plasmid pCR4-topoTA-RhoB was digested with SalI and EcoRI and the insert was cloned in the XhoI and EcoRI restriction sites of the plasmid pMG20-NTg3p-Nextein_K to obtain pMG20-N1-N2-Nextein_RhoB. The fusion protein NTg3p-NVDE_RhoB was expressed in E. coli strain BL21DE3 and purified by Ni-NTA chromatography according to standard methods (28).
c. Restoration of Phage Infecting Ability Through the Reconstitution of the Gene III
R3-CVDE-CTg3p fusion phages were incubated with the target complex NTg3p-NVDE_RhoB in the splicing buffer 18 h at 24° C. This mixture was added to an excess of E. coli XL1blue cells and after incubation at 37° C., aliquots were plated on 2YT-agar containing 100 μg/ml of ampicillin, 0.5% glucose. Phages recovering infecting ability were counted as colony forming units after overnight incubation at 37° C.
a. Phage Fusion Complex
The fragment hinge-Fc (aa: 226-447) of a human IgG1 has been amplified from the clone pBHuCgamma1 (30) with the primer pair 5′-TATATATGGATCCTGCCCACCGTGCCCAGCACCT-3′ (SEQ ID NO:27) and 5′-GCTAGTCAGTGCGGCCGCGAATTCTTTACCCGGAGACAGGGAGAG-3′ (SEQ ID NO:28) in order to insert NcoI, a stretch of six histidines and BglII in 5′ and NotI in 3′. The PCR product was digested and cloned in the NcoI and NotI restriction sites of the phagemid pCVDE-CTg3p vector providing the phagemid pCVDE-CTg3p-hinge-Fc (
a. Target Complex
Production of N1-N2-NVDE_K and coupling to protein A (spA). The fusion protein NTg3p-Nextein_K was expressed using the plasmid pMG20-N1-N2-NVDE_K in E. coli strain BL21DE3 and purified by Ni-NTA chromatography according to standard methods. NTg3p-Nextein_K was coupled with Protein A in molar ratio 1/1 on free primary amine (Lysin lateral chain) by the water soluble homo bifunctional glutaraldehyde. Coupling product was subjected to an IMAC purification procedure on a NiNTA Agarose resin (Qiagen) followed by a size exclusion gel chromatography (Amersham). The resulting complex was analysed by SDS PAGE and western blot.
b. Restoration of Phage Infecting Ability
The hinge-Fc-CVDE-CTg3p fusion phage were incubated with the target complex N1-N2-NVDE_hinge_Fc in the splicing buffer 18 h at 24° C. This mixture was added to an excess of E. coli XL1blue cells and after incubation at 37° C., aliquots were plated on 2YT-agar containing 100 μg/ml of ampicillin, 0.5% glucose. Phages recovering infecting ability were counted as colony forming units after incubation overnight at 3′7° C.
a. Phage Fusion Complex
A domain of variable heavy chain was isolated from a highly diversify antibody library (MutalBank-Millegen) through a screening against Klip-1 extracellular fragment. The VH-4K has a specific affinity against the Klip-1 extracellular fragment. The antibody fragment VH-4K was PCR amplified with the primers pair 5′-GCAGCCCCATAAACACACAGTATGT-3′ (SEQ ID NO:29) and 5′-ATATATATATGCGGCCGCGAATTCGAAGATCCGCCGCCAC-3′ (SEQ ID NO:30) in order to insert NotI in 3′ and cloned in the BglII and NotI restriction site of the phagemid pCVDE-CTg3p-hinge-Fc to replace the hinge-Fc with VH-4k to obtain pCVDE-CTg3p-VH-4k. Phage particles displaying the fusion complex VH-4k-CVDE-CTg3p on their surface were produced in the E. coli XL1blue harbouring pCVDE-CTg3p-VH-4k and co-infected with the hyperphage M13KO7ΔpIII (Progen). The phages were then prepared according to standard methods and the affinity to Klip1 protein was checked by ELISA.
b. Target Complex
Klip-1 extracellular fragment was PCR amplified from the vector pQE-31 (31) using the primer pair 5′-TATATACTCGAGGAAGAAAACATCCAGGGCGGAG-3′ (SEQ ID NO:31) and 5′-TATATATCTAGAAGGTCCATAGAGTTCACCTG-3′ (SEQ ID NO:32) was cloned into the vector pCR4-topoTA (Invitrogen). Klip-1 was removed from plasmid pCR4-topoTA-Klip-1 and cloned in the XhoI and EcoRI restriction sites of the plasmid pMG20-NTg3p-NVDE_K to obtain pMG20-NTg3p-NVDE_Klip1. The fusion protein NTg3p-NVDE_Klip1 was expressed in E. coli strain BL21DE3 and purified by Ni-NTA chromatography according to standard methods (28).
c. Restoration of Phage Infecting Ability
VH-4k-CVDE-CTg3p fusion phage were incubated with the target complex NTg3p-NVDE_Klip1 in the splicing buffer 18 h at 24° C. This mixture was added to an excess of E. coli XL1blue cells and after incubation at 37° C., aliquots were plated on 2YT-agar containing 100 μg/ml of ampicillin, 0.5% glucose. Phages recovering infecting ability were counted as colony forming units after incubation overnight at 37° C.
The two half parts of the intein (VDE) used in the fusion proteins of this example were N-VDE1-184 (amino acids 1 to 184) and C-VDE390-454 (amino acids 390 to 454).
a. Construction of the Target Complex
The N-terminal domain of the VDE (N-VDE: amino acids 1 to 184) were PCR amplified from pMG20-NTg3p-NVDE using the primer pair: MG587 5′-GAATTCCTGAAAGGTTGCTTTGCCAAGGGTACCAATGTTTTAATGGCGGA-3′ (SEQ ID NO:33) and MG588: 5′-AGTGCCGTTGCCGTTGCCGTTGTTTCTAGAATAAAGAATTGGAGCGTAAGTCTGGTAGG TA-3′ (SEQ ID NO:34) which allows to add EcoRI and XbaI restriction sites at the two extremities of the N-VDE and the linker SRNNGNGNT (SEQ ID NO:35) at the C-terminal part of the N-VDE.
The gene of MBP was amplified from pMALp2x (NEB) using the primer pair MG585 5′-TATCCATGGAAATCGAAGAAGGTAAACTGGTAATCT-3′ (SEQ ID NO:36) and MG586 5′-AGCAACCTTTCAGGAATTCTGAAATCCTTCCCTCGATCCCGAGGT-3′ (SEQ ID NO:37) which allows to add NcoI and EcoRI restriction sites at the two extremities of the MBP and the linker ISEFLK (SEQ ID NO:38) at the C-terminal part of the MBP. The FKBP2 gene was PCR amplified from a human placenta cDNA library (BD Bioscience) using the primer pair MG 525 5′-ATATATCTCGAGGGAGTGCAGGTGGAAACCATCT-3′ (SEQ ID NO:39) and MG 526 5′-TATATATGCGGCCGCTTATTCCAGTTTTAGAAGCTCCACATCGA-3′ (SEQ ID NO:40) which allows to add XhoI and Not/restriction sites at the two extremities of the FKBP2 and cloned into XhoI and NotI restriction sites of pMG20-NTg3P-NVDE (pMG54)to obtain the plasmid pMG54-FKBP.
The gene of FKBP2 was amplified from pMG54-FKBP using the primer pair MG589 s′-ACAACGGCAACGGCAACGGCACTAGAGGAGTGCAGGTGGAAACCATCTCCCCAGGA-3′ (SEQ ID NO:41) and MG590 5′-TATAAGCTTAGTGATGGTGATGGTGATGAGATCTGGATCCATAACTAGTTTCCAGTTTT AGAAGCTCCACATCGA-3′ (SEQ ID NO:42) which allows to add the linker GSRS (SEQ ID NO:43) at the N-terminal of FKBP2 and the hexa-histidine tag (6His), BamHI and HindIII restriction sites at the C-terminal of FKBP2.
The MBP and N-VDE PCR products were assembled through an overlap PCR to obtain PO41-1. N-VDE and FKBP2 PCR products were also assembled through an overlap PCR to obtain PO41-2. These PO41-1 and PO41-2 PCR products were then digested respectively with (Noel and XbaI) and (XbaI and HindIII) and cloned into NcoI and HindIII restriction sites of pTRC-His (Invitrogen) to obtain the plasmid pMG73-FKBP (
MBP2-ISEFLK-NVDE1-184-SRNNGNGNTR-FKBP2-GSRS-6His in E. coli strain BL21DE3 and purified by Ni-NTA and amylose chromatographies according to standard methods (28).
The N1-N2 domains of the gene III (NTg3p) was PCR amplified from pMG54-FKBP using the primer pair MG613 5′-TATAGAATTCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCAT TTACTAACGTCT-3′ (SEQ ID NO:44) and MG 609 5″-ATTGGTACCCTTGGCAAAGCAGCOACCCTCAGAACCGCCACCCTCAGAGCCGCCACCCT CAGAGCCGCCACCCTCAGAGCCGCCACCAGA-3′ (SEQ ID NO:45) which allows to add EcoRI in 5′ and KpnI in 3′ and cloned in the EcoRI and KpnI restriction sites of the pMG73-FKBP to generate the plasmid pMG76L-FKBP (
b. Phage Fusion Complex
The C-terminal domain (C-VDE: amino acids 390 to 454) of the VDE was amplified from pCVDE-CTgP3p using the primer pair MG593: 5′-AGCGAATTCACTAGTGTTTTGCTTAACGTTCTTTCGA-3′(SEQ ID NO:46) and MG594: 5′-TATGGATCCGTCCTCCTTCTCGTCGCAATTGTGGACGACAACCTGGTT (SEQ ID NO:47) which allows to add Spe I and BamBI restriction sites at the two extremities of the C-VDE and the linker CDEKEDGS (SEQ ID NO:48) at the C-terminal part of the C-VDE.
The FRB gene was PCR amplified from a human placenta cDNA library (BD Bioscience) using the primer pair MG—523 5′-ATATATAGGATCCGCAGAGCTGATCCGAGTGGCCATC-3TMQ ID NO:49) and MG524 5′-TATATATGCGGCCGCGAATTCCTGCTTTGAGATTCGTCGGAACACAT-3′ (SEQ ID NO:50) which allows to add BamHI and NotI restriction sites at the two extremities of the FRB and cloned into BamHI and NotI restriction sites of pMG64 (pCVDE-CTgP3p) to obtain pMG64-FRB.
The FRB gene was amplified from pMG64-FRB using the primer pair MG591 5′-TCAGTCTAGAATCCTCTGGCATGAGAT-3′(SEQ ID NO:51) and MG592 5′-GGACTAGTCTTTGAGATTCGTCGGAACACATG-3′(SEQ ID NO:52) which allows to add EcoRI and BamHI restriction sites at the two extremities of the FRB.
The FRB and C-VDE PCR products were digested respectively with (EcoRI and SpeI) and (SpeI and BamHI) and then cloned into EcoRI and BamHI restriction sites of pMG73-FKBP to obtain the plasmid pMG73-FRB (
This vector allowed to express the fusion protein MBP2-ISEFGSSR-FRB-TS-CVDE390-454-CDEKEDGSRS-6His in E. coli strain BL21DE3 and purified by Ni-NTA and MBP chromatographies according to standard methods (28).
The C-Terminal of protein III (gene III) of bacteriophage M13 was PCR amplified and cloned in the pMG73-FRB plasmid to replace at the C-terminal part of the C-VDE both the linker DEKEDGSRS and the 6His-tag with the end of G2 PIII(GSGGGS) and CTg3p in order to restore a native PII after splicing.
The fragment FRB-CVDE was PCR amplified from pMG73-FRB using the primer pair MG591: 5′-TCAGTCTAGAATCCTCTGGCATGAGAT-3′ (SEQ ID NO:53) and MG612 5′-ATAGGATCCGCAATTGTGGACGACAACCTGGTTGGCAAGCA-3′ (SEQ ID NO:54) which allows to remove the linker DEKED and keep BamHI at the C-terminal part of the CVDE390-454.
The CT of the gene III (CTg3p) was amplified from pMG64-FRB using primer pair 5′-TATAGGATCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAAC-3′ (SEQ ID NO:55) and 5′-CCCAAGCTTTTAAGACTCCTTATTACGCAGTATGTTAGC-3′ (seq ID NO:56) which allows to add BamHI in 5′ and HindIII in 3′ of the extremities of CTg3p.
The FRB-CVDE390-454 and CTg3p PCR products were digested respectively with (XbaI and BamHI) and (BamHI and Hind III) and then cloned into XbaI and BamHI restriction sites of pMG73-FRB providing the plasmid pMG77-FRB (
The fusion protein MBP2-ISEFGSSR-FRB-TS-CVDE390-454-GSGGGS-CTg3p was expressed in E. coli strain BL21DE3 and purified by Ni-NTA and amylose chromatographies according to standard methods (28).
The insert FRB-CVDE390-454-CTPIII from pMG77-FRB was cloned in ApaI and NdeI restriction sites of the phagemid pMG64-FRB to obtain pMG78-FRB (
This vector allowed to express in “display” on a phage membrane the fusion protein FRB-TS-CVDE390-454-GSGGGS-CTg3p with a signal sequence pelB and under the control of a Lac promoter.
c. Reconstitution of the Gene III Protein Via Trans-Splicing Following Protein-Target Interaction
The pair of heterodimerisation domains, FKBP and FRB of the two fusion proteins allow a tight ternary complex formation in presence of rapamycin. The dissociation constant (Kd) of the FKBP-rapamycin-FRB complex was reported to be 2 nM (32). Thus, the protein trans-splicing assay was triggered by the addition of the rapamycin.
Four pairs of purified fusion proteins were incubated with or without rapamycin (10 μM), 2-3 h at 25° C. or 30° C. in the assay buffer (50 mM Tris-HCl pH-7, 300 mM NaCl, 1 mM EDTA, 10% glycerol, 2 mM DTT) (
Trans-splicing assays were also performed using a combination of purified constructs with or without the N- or C-terminal fusion part of the g3p (
Negative controls were performed using the purified fusion protein 1 alone without or with rapamycin (
No splice products were detected in the absence of rapamycin (
The reconstitution of the protein III was confirmed by Western blotting using the antibodies directed against the N-(anti-MBP antibody, NEB) and C-terminal (anti-pIII antibody, PSKAN3, MoBiTec) of the expected MBP-g3p (splicing product 2) at 85.5 Kda (
d. Restoration of the Phage Infecting Ability Through the Reconstitution of the Gene III
The phagemid pMG78-FRB encodes the FRB protein as an N-terminal fusion of C-terminal domain of (the intein (CVDE) and the C-terminal domain of the pIII. Phage particles displaying the FRB on their surfaces were produced in the E. coli XL1blue harbouring the plasmid pMG78-FRB and co-infected with the hyperphage M13KO7ΔpIII (Progen). The phages were then prepared according to standard methods (28).
The fusion target complex MBP2-ISEF-N1G1N2-(GGGSGGGSGGGSEGGGSEGGGSEGGGSEGG)-NVDE1-184-SRNNGNGNTR-FKBP2-GSRS-6His (2-5 μM) and the phage displaying the fusion protein FRB-TS-CVDE390-454-GSGGGS-CTg3p (108-1010 phages per assay) were incubated, with or without rapamycin (10 μM), 3 h at 25° C. or 30° C. in the assay buffer (50 mM Tris-HCl pH=7, 300 mM NaCl, 1 mM EDTA, 10% glycerol, 2 mM DTT).
This mixture was added to an excess of E. coli XL1blue cells and after incubation at 37° C., aliquots were plated on 2YT-agar containing 100 μg/ml of ampicillin, 1% glucose and incubated overnight at 37° C. The ratio phage/bacteria were set up in order to minimize the non specific infection. The phages recovering infecting ability counted as colony forming units was substantially higher (100 times) in the presence of rapamycin than without rapamycin. Furthermore, a negative control without the fusion target complex showed few background clones.
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
TFP1 gene product to the 69 kD subunit of the vacuolar H+-adenosine triphosphate. Science, 250, 651-657.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06290126.9 | Jan 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2007/000128 | 1/18/2007 | WO | 00 | 7/16/2008 |
