Patent Application
20120070845
This invention relates to methods, systems and reagents for improved immunodetection of expressed recombinant proteins. Specifically this invention relates to the generation of new epitope tags and to the generation of novel antibodies against these epitope tags in addition to already existing epitope tags.
Epitope tagging has become an important tool for detecting, localizing and purifying expressed recombinant proteins (Nygren et al., Trends Biotechnol. 12: 184-188 (1994)). This methodology involves the fusion of the tag amino acid sequence to the amino or carboxy terminus of a protein of interest, and then identifying the tag with a monoclonal antibody (mAb). For most biochemical applications, the use of epitope tags eliminates the need to generate an antibody to the specific protein that is to be detected and/or purified.
Currently, there are several validated mAb epitopes in wide use for protein tagging, including the FLAG peptide epitope (8 amino acid residues reactive to mAbs M1 and M2) (Hopp et al., BioTechnology 6: 1204-1210 (1988)), the V5 epitope (14 amino acids found in the P and V proteins of paramyxovirus, Simian Virus 5) (Southern et al., J Gen Virol. 72: 1551-1557 (1991)), the myc epitope (10 amino acids derived from the c-myc proto-oncogen product) (Evan et al., Mol. Cell. Biol. 5: 3610-3616 (1985)), the HA epitope (9 amino acids from the hemagglutinin of influenza virus) (Wilson et al., Cell 37(3):7 67-778 (1984)), and the 6× His epitope (Lindner et al., BioTechniques 22(1): 140-149 (1997)).
However, the choice of an epitope tag depends on the application, because not all tags and in particular the corresponding mAbs are equally suitable for all immunodetection methods, e.g. Western blotting, immunofluorescence staining, immunoprecipitation, and flow cytometry. Accordingly, there is a need in the art for novel detection tags and the antibodies recognizing those detection tags.
Accordingly, the present invention fulfills this need by providing in one aspect epitope tags and antibodies against these tags. These epitope tags may comprise an amino acid sequence of SEQ ID NO: 1 (SGFANELGPRLMGK). A nucleic acid molecule is also provided wherein the nucleic acid molecule encodes the amino acid SEQ ID NO: 1. In one embodiment, the nucleic acid sequence is identical to SEQ ID NO: 2.
The antibodies selectively bind SEQ ID NO: 1. In addition, antibodies that selectively bind SEQ ID NO: 3 are also provided. Compared to already existing antibodies, the antibodies described herein exhibit an increased affinity to epitope tags comprising SEQ ID NO: 1 and SEQ ID NO: 3. In some embodiments, the antibodies are rat IgG monoclonal antibodies (mAb).
In another aspect, fusion proteins and methods for making these proteins are provided. The fusion proteins comprise an epitope tag comprising an amino acid sequence SEQ ID NO: 1 and an amino acid sequence of a target protein.
In yet another aspect, a method of making a fusion protein comprising SEQ ID NO: 1, and a target protein is provided. The method comprises introducing into a host cell a nucleic acid sequence comprising a first part encoding SEQ ID NO: 1, and a second part encoding the amino acid sequence for the target protein; culturing the host cell under conditions whereby the fusion protein is expressed; isolating the fusion protein by binding the fusion protein to an antibody that binds SEQ ID NO: 1 or SEQ ID NO: 2.
In yet another aspect, kits for diagnostic assays for detecting and analyzing fusion proteins comprising SEQ ID NO: 1, or SEQ ID NO: 3 are provided. These kits may comprise an antibody that binds to SEQ ID NO: 1, or SEQ ID NO: 3, detection means such as another antibody, and, optionally a label.
In another aspect, a cloning vector comprising a nucleic acid sequence encoding SEQ ID NO: 1 is provided. In some embodiment, the nucleic acid sequence comprises SEQ ID NO: 2. The cloning vector may be provided as a kit. Preferably, the kit may also include a OLLAS-tagged control protein construct DNAs.
Monoclonal antibodies against epitope tags are an efficient, convenient and rapid method for detecting recombinant protein expression (Jarvik and Telmer, Annu Rev Genet. 32:601-618 (1998)). If there is no antibody against the protein of interest, adding an epitope tag to this protein allows for protein detection with an antibody against the epitope sequence. Accordingly, in one aspect, novel epitope tags and antibodies against these tags are disclosed.
As used herein, the term “epitope” or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond or a site on a molecule against which an antibody will be produced and/or to which an antibody will bind. For example, an epitope can be recognized by an antibody defining the epitope. An epitope can be either a “linear epitope” (where a primary amino acid primary sequence comprises the epitope; typically at least 3 contiguous amino acid residues, and more usually, at least 5, and up to about 8 to about 10 amino acids in a unique sequence) or a “conformational epitope” (an epitope wherein the primary, contiguous amino acid sequence is not the sole defining component of the epitope). A conformational epitope may comprise an increased number of amino acids relative to a linear epitope, as this conformational epitope recognizes a three-dimensional structure of the peptide or protein. For example, when a protein molecule folds to form a three dimensional structure, certain amino acids and/or the polypeptide backbone forming the conformational epitope become juxtaposed enabling the antibody to recognize the epitope. Methods of determining conformation of epitopes include but are not limited to, for example, x-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy and site-directed spin labeling and electron paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996), the disclosure of which is incorporated in its entirety herein by reference.
In one embodiment, the epitope tag may comprise a 14 amino acid sequence presented herein as SEQ ID NO: 1: SGFANELGPRLMGK, which may be referred to as OLLAS. This sequence resides in the junction between a highly flexible domain in E. coli OmpF protein, named OFL (OmpF linker), and a mouse Langerin extracellular domain (mLangerin ECD). These epitope tags are referred to herein as OLLAS (E. coli OmpF Linker and mouse Langerin fusion Sequence epitope.)
Alternatively, the epitope tag may comprise a fragment of SEQ ID NO: 1. Some suitable examples of fragments include, but are not limited to, an amino acid sequence with up to 4 amino acid deletion or changes at the N-terminal of the amino acid of SEQ ID NO: 1 or an amino acid sequence with up to 2 amino acid deletion or changes at the C-terminal of the amino acid of SEQ ID NO: 1. In different embodiments, the suitable fragment comprises at least one of the following:
In another embodiment, an isolated nucleic acid sequence encoding SEQ ID NO: 1 is provided. One with ordinary skills in the art will undoubtedly understand that different nucleic acid sequences may encode the same amino acid sequence. One example is presented herein as SEQ ID NO: 2: AGT GGC TTT GCG AAT GAA TTG GGA CCT AGG TTG ATG GGC AAG. Furthermore, isolated nucleic acid sequences encoding fragments of SEQ ID NO: 1 are provided. Sequences for different fragments can be easily construed by one with ordinary skill in the art.
The epitope tags of the instant invention and the nucleic acid sequences encoding those epitopes may be obtained by a variety of methods well known in the art. Considering short size of these amino- and nucleic acid sequences, one of the most convenient ways to make these molecules is to order them from suppliers who synthesize short peptides and oligonucleotides. Suitable examples of such companies are Invitrogen, Inc., (Carlsbad Calif.) and The Midland Certified Reagent Company, Inc. (Midland, Tex.).
Antibodies against the OLLAS epitope tags are also provided. Such antibodies, referred to as OLLA-2, selectively bind to SEQ ID NO: 1 and its fragments.
In some embodiments, these antibodies may exhibit an increased affinity to the OLLAS epitope tags compared to other available antibodies and, thus, may enhance the performance of a variety of immunodetection methods, including, but not limited to, Western Blot, immunocytochemistry, immunohistochemistry, flow cytometry, and immunoprecipitation. It has been shown that, by Western blotting, 1 μg/ml of anti-V5 mAB is capable of detecting OLLAS in cell lysates from about 1250 cells transfected with SEQ ID NO: 1 and 0.5 μg/ml of anti-Flag mAB M2 is capable of detecting OLLAS in cell lysates from about 15,000 such cells. Accordingly, preferably OLLA-2 may be capable of detecting less than the amount detected by anti-Flag mAB M2 and anti-V5 mAB. More preferably, about 1 μg/ml of OLLA-2 may be capable of detecting OLLAS from about 10 cells, and 0.5 μg/ml of OLLA-2 may be capable of detecting OLLAS from about 78 of these cells.
In another embodiment, antibodies against a known epitope, FLAG, are provided which are herein referred to as the new anti-FLAG L5. The FLAG epitope comprises an 8 amino acid sequence SEQ ID NO: 3: DYKDDDDK. Preferably, new anti-FLAG L5 is capable of binding to SEQ ID NO: 3 with increased affinity compared to existing antibodies against FLAG, and thus, enhances the performance of a variety of immunodetection methods, including Western Blot, immunocytochemistry, immunohistochemistry, flow cytometry, and immunoprecipitation. In one embodiment, the new anti-FLAG L5 may detect both N-terminal and C-terminal FLAG tagged proteins about 2 to 8 times better than the conventional anti-FLAG mAb M2 using the Western blot analysis. Preferably, in Western blot analysis, 1 mg/ml of new anti-FLAG L5 can detect less than 100 ng of N-terminally FLAG tagged purified protein or lysates from about 2,500 cells transfected with N-terminally FLAG tagged recombinant protein construct, and more preferably it can detect about as little as 25 ng of N-terminally FLAG tagged purified protein or lysates from about 1,250 cells transfected with N-terminally FLAG tagged recombinant protein construct.
OLLA-2 and new anti-FLAG L5 antibodies may take one of many forms known in the art. The term “antibody” is used here in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody derivatives, functional equivalents and antibody fragments so long as they retain its antigen binding capability, generally including, but not limited to, the specific binding member or antigen-binding portion. The terms “specific binding member” and “antigen-binding portion” include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody (v) a dAb fragment, which comprises a VH domain; (vi) an isolated complementarily determining region (CDR); (vii) a ‘scAb’, an antibody fragment containing VH and VL as well as either CL or CH; and (viii) artificial antibodies based upon protein scaffolds, including but not limited to fibronectin type III polypeptide antibodies (e.g., see U.S. Pat. No. 6,703,199, issued to Koide on Mar. 9, 2004 and PCT International Application Publication No. WO 02/32925).
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies may be generated from different species, including but not limited to, mice, rats, rabbits, or primates.
For example, the monoclonal antibodies to be used in accordance with the methods disclosed herein may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256, 495-497 (1975), which is incorporated herein by reference. Alternatively, the monoclonal antibodies may be made by recombinant DNA methods which are disclosed, for example, in U.S. Pat. No. 4,816,567, which is incorporated herein by reference, and are also generally described below. Finally, techniques are available to the artisan for the selection of antibody fragments from libraries using enrichment technologies, including but not limited to phage display, ribosome display (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou, et al., 1997, Nature Biotechnology 15: 29-34) and/or yeast display (Kieke, et al., 1997, Protein Engineering 10: 1303-1310.)
Beyond species specific monoclonal antibodies described above, the antibodies of the present invention may also be in the form of a “chimeric antibody”, a monoclonal antibody constructed from the variable regions derived from say, the murine source, and constant regions derived from the intended host source (e.g., human; for a review, see Morrison and Oi, 1989, Advances in Immunology, 44: 65-92). The variable light and heavy genes from the rodent (e.g., mouse) antibody are cloned into a mammalian expression vector which contains an appropriate human light chain and heavy chain coding region, respectively. These heavy and light “chimeric” expression vectors are cotransfected into a recipient cell line and selected and expanded by known techniques. This cell line may then be subjected to known cell culture techniques, resulting in production of both the light and heavy chains of a chimeric antibody Such chimeric antibodies have historically been shown to have the antigen-binding capacity of the original rodent monoclonal while significantly reducing immunogenicity problems upon host administration.
A logical improvement to the chimeric antibody is the “humanized antibody,” which arguably reduces the chance of the patient mounting an immune response against a therapeutic antibody when compared to use of a chimeric or full murine monoclonal antibody The strategy of “humanizing” a murine Mab is based on replacing amino acid residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grafting of entire complementarily determining regions (Jones et al., 1986, Nature 321: 522-526). This technology is again now well known in the art and is represented by numerous strategies to improve on this technology; namely by implementing strategies including, but not limited to, “reshaping” (see Verhoeyen, et al., 1988, Science 239: 1534-1536), “hyperchimerization” (see Queen, et al., 1991, Proc. Natl. Acad. Sci. 88:2869-2873) or “veneering” (Mark, et al., 1994, Derivation of Therapeutically Active Humanized and Veneered anti-CD18 Antibodies Metcalf end Dalton, eds. Cellular Adhesion: Molecular Definition to Therapeutic Potential. New York: Plenum Press, 291-312). These strategies all involve to some degree sequence comparison between rodent and human sequences to determine whether specific amino acid substitutions from a rodent to human consensus is appropriate. Whatever the variations, the central theme involved in generating a humanized antibody relies on CDR grafting, where these three antigen binding sites from both the light and heavy chain are effectively removed from the rodent expressing antibody clone and subcloned (or “grafted”) into an expression vector coding for the framework region of the human antibody. Therefore, a “humanized antibody” is effectively an antibody constructed with only murine CDRs (minus any additional improvements generated by incorporating one or more of the above mentioned strategies), with the remainder of the variable region and all of the constant region being derived from a human source.
Yet another improvement over re-engineered antibodies as reviewed above is the generation of fully human monoclonal antibodies. The first involves the use of genetically engineered mouse strains which possess an immune system whereby the mouse antibody genes have been inactivated and in turn replaced with a repertoire of functional human antibody genes, while leaving other components of the mouse immune system unchanged. Such genetically engineered mice allow for the natural in vivo immune response and affinity maturation process which results in high affinity, fully human monoclonal antibodies This technology is again now well known in the art and is fully detailed in various publications, including but not limited to U.S. Pat. Nos. 5,939, 598; 6,075,181; 6,114,598; 6,150,584 and related family members (assigned to Abgenix, disclosing their XenoMouse technology); as well as U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877, 397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429 (assigned to GenPharm International and available through Medarex, under the umbrella of the “UltraMab Human Antibody Development System”). See also a review from Kellerman and Green (2002, Curr. Opinion in Biotechnology 13: 593-597).
In the preferred embodiments, the L2 anti-OLLAS antibodies (which may also be referred to as OLLA-2 antibodies) and new anti-FLAG L5 antibodies are rat monoclonal antibodies.
In another aspect, fusion proteins are provided. In one embodiment, the fusion proteins may comprise epitope tags encoded by SEQ ID NO: 1 or its fragments (such as SEQ ID NO: 4-SEQ ID NO: 17), and an amino acid sequence of a target protein. The epitope tags may be connected to the C-terminal, the N-terminal or both N- and C-termini of the target protein. Furthermore, methods of making such fusion proteins are provided. The methods comprise introducing into a host cell a nucleic acid sequence comprising a first part encoding the epitope tag, and a second part encoding an amino acid sequence for the target protein, culturing the host cell under conditions that promote expression of protein linked to the epitope tag, and isolating the fusion protein.
The term “isolating” or “isolated” is used herein as it is used within the art. It means that antibodies, antibody fragment/specific binding members, nucleic acid molecules, and proteins are free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology (practiced ill vitro) or in vivo. “Isolating” or “isolated” covers any form containing the identified and characterized antibodies, antibody fragments/specific binding members, nucleic acid molecules, proteins described herein following their removal from that initial environment.
Techniques for such manipulations are well known and are readily available to the artisan of ordinary skill in the art. Many treatises on recombinant DNA methods have been published, including Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, (1988), Davis, et al., Basic Methods in Molecular Biology, Elsevier (1986), and Ausubel, et al., Current Protocols in Molecular Biology, Wiley Interscience (1988). These reference manuals are specifically incorporated by reference herein in their entirety.
In general, a nucleic acid molecule comprising a sequence encoding an epitope tag and a nucleic acid molecule encoding a target protein may be linked, in whole or in part, to form “recombinant DNA molecules” which encode a desired fusion protein. These recombinant DNA molecules may be prepared using any known technique. For example, the sequences for the eptiope tag and the target protein may be cloned, sequenced and ligated to make the recombinant DNA molecule for the fusion protein. In one embodiment, the sequences may be generated and cloned using the polymerase chain reaction (PCR). Alternatively, a cloning vector comprising of DNA or RNA may be employed. Cloning vectors are defined herein as an agent that can carry and reproduce a DNA fragment into a host cell. For most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA. It is within the purview of the skilled artisan to determine an appropriate vector for a particular gene transfer and generation of a recombinant DNA for a fusion protein or other use.
In one aspect, cloning vectors comprising a nucleic acid sequence encoding SEQ ID NO: 1 are provided. As stated above, cloning vectors are defined herein as agents that can carry and reproduce a DNA fragment into a host cell. In one exemplary embodiment, the nucleic acid sequence may comprise SEQ ID NO: 2. The cloning vector may be prepared by any known technique such as described in reference manuals cited above. The cloning vector may further include a polyadenylation signal, a transcription termination sequence, and a multiple cloning site comprising at least one endonuclease restriction site. The vector may further comprise a promoter. In some embodiments, in the operational vector, the nucleic acid sequence may be located upstream of the polyadenylation signal and the transcription termination sequence, and the at least one endonuclease restriction site may be located upstream of said polyadenylation signal and the transcription termination sequence.
A target nucleic acid sequence may be cloned into the multiple cloning site of the cloning vector. It is preferable that the nucleic acid sequence encoding SEQ ID NO: 1 or its fragments, and the target nucleic acid sequence may be transcribed as a single mRNA and translated into a single amino acid sequence that includes SEQ ID NO: 1 or its fragments. Depending on the desired location of the epitope tag in the fusion protein, the at least one endonuclease restriction site may be located downstream of the nucleic acid sequence encoding SEQ ID NO: 1 or its fragments for N-terminal tagging, or upstream of the nucleic acid sequence encoding SEQ ID NO: 1 or its fragments for C-terminal tagging, or both.
The cloning vector may be provided as a kit. In addition to the cloning vector, the kit may include a forward primer; a reverse primer; a ligase; at least one restriction endonuclease; an aliquote of competent cells; instructions or any combinations thereof. The kit may also include a control. In one exemplary embodiment, the control may be a vector comprising a DNA sequence that encodes a known OLLAS-tagged protein (e.g., a GFP protein).
Once recombinant DNA molecules are prepared, they may be inserted into an expression vector and transfected into a host cell. Methods of subcloning nucleic acid molecules of interest into expression vectors, transforming or transfecting host cells containing the vectors, and methods of making substantially pure protein comprising the steps of introducing the respective expression vector into a host cell, and cultivating the host cell under appropriate conditions are well known. The fusion protein so produced may be harvested from the host cells in conventional ways. Any known expression vector may be utilized to practice this portion of the invention, including any vector containing a suitable promoter and other appropriate transcription regulatory elements. The resulting expression construct is transferred into a prokaryotic or eukaryotic host cell to produce recombinant protein.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency.
Commercially available mammalian expression vectors which may be suitable, include but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIanp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565). Also, a variety of bacterial expression vectors are available, including but not limited to pCR2.1 (Invitrogen), pET1 la (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungal cell expression vectors may be used, including but not limited to pYES2 (Invitrogen) and Pichie expression vector (Invitrogen). Also, a variety of insect cell expression vectors may be used, including but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of bovine, porcine, monkey and rodent origin; and insect cells. Mammalian species which may be suitable, include but are not limited to, L cells L-M(TK−) (ATCCCCL1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCCHTB-85), 293 (ATCCCRL1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL1650), COS-7(ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1(ATCC CCL 26), MRC-5 (ATCCCCL171) and CPAE (ATCC CCL 209).
The fusion proteins of interest may be isolated using any known protein purification technique. Suitable examples include, but are not limited to, immuno-affinity chromatography, affinity chromatography, protein precipitation, buffer exchange, ion exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, electrophoresis or combination thereof. In the preferred embodiment, the fusion protein is isolated using the OLLA-2 or new anti-FLAG L5 antibodies. These antibodies may be dissociated from the fusion protein using any known techniques such as by cleaving the antibody off using a protease or washing the antibody-antigen complex with a solution which favors dissociation of the antigen and the antibody (for example, using the solution of high ionic strength). Many of these techniques are described, for example, in Biochemistry, R. H. Garrett and C. M. Grisham, Suanders College Publishing, (1995); and Molecular and Cell Biology, Third Edition, H Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, J. Darnell, Scientific American Books, Inc. (1995), which are incorporated herein by reference.
In yet another aspect, kits for diagnostic assays for detecting and analyzing tagged protein are provided. Such assays may be carried out by any techniques known and available to the artisan, including but not limited to Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. These kits may comprise any of the antibodies described above, detection means corresponding to the antibody, and, optionally a label.
The antibodies described herein may be used as the basic reagents in a number of different immunoassays to determine the presence of a protein tagged either with OLLAS or FLAG epitopes in a sample. Generally speaking, the antibodies can be employed in any type of immunoassay, whether qualitative or quantitative. This includes both the two-site sandwich assay and the single site immunoassay of the non-competitive type, as well as in traditional competitive binding assays.
One embodiment of interest, for ease of detection, and its quantitative nature, is the sandwich or double antibody assay, of which a number of variations exist, all of which are intended to be encompassed by this portion of the present invention. For example, in a typical forward sandwich assay, unlabeled antibody is immobilized on a solid substrate, e.g., microtiter plate wells, and the sample to be tested is brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of inducing a detectable signal, is then added and incubation is continued allowing sufficient time for binding with the antigen at a different site and the formation of a ternary complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, which may be quantitated by comparison with a control sample containing known amounts of antigen.
Variations on the forward sandwich assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse sandwich assay in which the labeled antibody and sample to be tested are first combined, incubated and added to the unlabelled surface bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. As used herein, “sandwich assay” is intended to encompass all variations on the basic two-site technique.
For the sandwich assays, the only limiting factor is that both antibodies have different binding specificities for the OLLAS or FLAG epitopes. Thus, a number of possible combinations are possible. As a more specific example, in a typical forward sandwich assay, a primary antibody is either covalently or passively bound to a solid support. The solid surface is usually glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surfaces suitable for conducting an immunoassay. The binding processes are well known in the art. Following binding, the solid phase-antibody complex is washed in preparation for the test sample. An aliquot of the sample containing a protein tagged with either OLLAS or FLAG epitope to be tested is then added to the solid phase complex and incubated at 25° C. for a period of time sufficient to allow binding of any tagged proteins present to the antibody specific for that tag. The second antibody is then added to the solid phase complex and incubated at 25° C. for an additional period of time sufficient to allow the second antibody to bind to the primary antibody-antigen solid phase complex. The second antibody may be linked to a reporter molecule, the visible signal of which is used to indicate the binding of the second antibody to any antigen in the sample. By “reporter molecule”, as used in the present specification is meant a molecule which by its chemical nature, provides an analytically detectable signal which allows the detection of antigen-bound antibody. Detection must be at least relatively quantifiable, to allow determination of the amount of antigen in the sample, this may be calculated in absolute terms, or may be done in comparison with a standard (or series of standards) containing a known normal level of antigen.
The most commonly used reporter molecules in this type of assay are either enzymes or fluorophores. In the case of an enzyme immunoassay an enzyme is conjugated to the second antibody, often by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are well known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-antigen complex and allowed to bind to the complex, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the tertiary complex of antibody-antigen-labeled antibody. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an evaluation of the amount of antigen that is present in the serum sample.
Additionally, fluorescent compounds, such as fluorescein or rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope. As in the enzyme immunoassay (EIA), the fluorescent-labeled antibody is allowed to bind to the first antibody-tagged protein complex. After washing the unbound reagent, the remaining ternary complex is then exposed to light of the appropriate wavelength, and the fluorescence observed indicates the presence of the antigen. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.
In another embodiment, the sample to be tested may be used in a single site immunoassay wherein it is adhered to a solid substrate either covalently or noncovalently. An unlabeled anti-OLLAS or anti-FLAG antibody is brought into contact with the sample bound on the solid substrate. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex a second antibody, labeled with a reporter molecule capable of inducing a detectable signal, is then added and incubation is continued allowing sufficient time for the formation of a ternary complex of antigen-antibody-labeled antibody. For the single site immunoassay, the second antibody may be a general antibody (i.e., zenogeneic antibody to immunoglobulin, particularly anti-(IgM and IgG) linked to a reporter molecule) that is capable of binding an antibody that is specific for the tagged protein of interest.
Commercially available, conventional anti-tag mAbs are mostly, if not all, made from mouse. The mouse anti-tag mAbs are not readily usable for the studies on mouse tissues or in vivo applications in mouse, unless they are directly labeled with fluorochromes, enzymes, or other detectable means, which would make those materials rare and costly. Therefore, the new anti-OLLAS tag mAb OLLA-2 from rat IgG will provide an extra advantage over conventional mouse anti-tag mAbs, as the inventors have illustrated that mAb OLLA-2 can be used directly in mouse in vivo and detected in mouse tissues without special, direct labelings.
Selected embodiments will not be further discussed in the following example. The example is illustrative only, and is not intended to limit the instant disclosure in any way.
Materials and Methods
Production of Epitope Specific MAb hybridoma.
Animals and cell lines: Wistar Furth rats were purchased from Charles River Laboratories (Wilmington, Mass.). C57BL/6 and BALB/c mice were purchased from Taconic Farms (Hudson, N.Y.) and Charles River Labs, and used at 6-8 wks of age. All animals were maintained under specific pathogen-free conditions. Animal care and experiments were conducted according to institutional guidelines of the Rockefeller University and Memorial Sloan-Kettering Cancer Center.
Chinese hamster ovary (CHO) cells and 293T cells were cultured in DMEM with 7% FBS or 5% Ultra-Low IgG FBS supplemented with 2mM glutamine (Gibco BRL, Invitrogen, Carlsbad, Calif.), antibiotics (Invitrogen) and non-essential amino acids (Invitrogen).
MAb hybridoma production: Generation and screening of mAb hybridomas against FLAG/OFL tagged mLangerin ECD were described previously (Cheong et al., J Immunol Methods. 2007 324(1-2):4842 (2007)). In brief, Wistar Furth rats were immunized with the purified proteins of mLangerin ECD fused with OFL and human IgG1 Fc (GenBank accession no. DQ917567, SEQ ID NO: 25) and FLAG/OFL tagged mLangerin ECD (GenBank accession no. DQ917568, SEQ ID NO: 26), followed by the injection of mouse dendritic cells enriched from ear skin organ cultures (Cheong et al., 2007). Then, the cells from spleen were used for hybridoma fusion at the Monoclonal Antibody Core Facility of the Rockefeller University and Memorial Sloan Kettering Cancer Center. Culture supernatants from the hybridomas were screened on ELISA with FLAG/OFL tagged mLangerin ECD protein as previously described (Cheong et al., 2007).
Reagents: To purify mAbs L2, L5, L31 and NLDC145, the supernatants of individual hybridomas were cultured and the mAbs were purified with protein G (Pierce, Rockford, Ill.; GE Healthcare, Piscataway, N.J.) column, according to the manufacture's instructions. The following reagents were purchased; ANTI-FLAG® M2, DAPI (Sigma-Aldrich, St. Louis, Mo.), anti-beta actin (Abcam Inc., Cambridge, Mass.), anti-human CD8 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), pEGFP-N1, anti-GFP (Clontech, Mountain View, Calif,), anti-B220 (BD Biosciences, San Jose, Calif.), anti-V5 (Invitrogen), HRP-conjugated anti-mouse IgG and HRP- or PE-conjugated anti-rat IgG (Southern Biotech, Birmingham, Ala.), PE-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.), Alexa350-, Alexa488- or Alexa647-conjugated anti-rat IgG and Alexa647-conjugated anti-mouse IgG (Molecular Probes, Invitrogen).
Vector constructions and expression of recombinant proteins: The construction, expression and purification of murinized heavy and light chains for anti-DEC205 mAb NLDC145 (mNLDC145) and isotype control mAb have been described (Hawiger et al., J Exp Med. 194(6):769-779 (2001)). The sequences of OLLAS peptide (SGFANELGPRLMGK/SEQ ID NO: 1; GenBank accession no. EF635496, SEQ ID NO: 27) or OLLAS-tagged ovalbumin (GenBank accession no. EF635488, SEQ ID NO: 28) were inserted at the carboxy-terminus in heavy chains of mNLDC145 or isotype control mAbs. 293T cells were transfected with the expression vectors for mNLDC145 or isotype control mAbs carrying OLLAS-tagged inserts. Then, the culture supernatants or mAbs purified by Protein G affinity column were used for further analyses. Soluble FLAG-mLangerin.ECD protein was purified from culture supernatants of the stable CHO/FLAG-mLangerin.ECD cells by ANTI-FLAG® M1 Affinity Gel (Sigma-Aldrich) following the manufacturer's instruction.
The generation of expression vectors for the open reading frames (ORFs) of full-length mLangerin and soluble fusion mLangerin ECD with FLAG/OFL was described previously (Cheong et al., 2007). The cDNAs for epitope tags or subdomains of individual genes were generated by PCR, sequenced, and ligated to make the ORFs for respective recombinant fusion proteins. Then, the ORFs were cloned into pCMV mammalian expression vector (Clontech) and stably transfected into CHO cells or transiently transfected to 293T cells. The expression vectors for the ORFs of following recombinant proteins were generated; FLAG-mLangerin.ECD (GenBank accession no. EF635489, SEQ ID NO: 29), FLAG-OFL/2-mLangerin.ECD (GenBank accession no. EF635490, SEQ ID NO: 30), FLAG-mSIGN-R1.ECD (GenBank accession no. EF635491, SEQ ID NO: 31), rat P11-FLAG.HA (GenBank accession no. EF635492, SEQ ID NO: 32), human CD8.ECD-OFL-mLangerin.ECD (GenBank accession no. EF635493, SEQ ID NO: 33), hCD8.ECD-OFL-mLangerin.ECD.deletion.#1 (GenBank accession no. EF635494, SEQ ID NO: 34), hCD8.ECD-OFL-mLangerin.ECD.deletion.#2 (GenBank accession no. EF635495, SEQ ID NO: 35), OT1-EGFP-OLLAS (GenBank accession no. EF635497, SEQ ID NO: 36), FLAG.OLLAS-EGFP (GenBank accession no. EF635498, SEQ ID NO: 37). V5-hIgG1Fc-OLLAS was generated from V5-hIgG1Fc-OFL-mLangerin.ECD (GenBank accession no. DQ917567, SEQ ID NO: 25; Cheong et al., 2007) by replacing OFL-mLangerin.ECD with OLLAS. OLLAS-EGFP and mSIGN-R1.Cytosol-OLLAS-EGFP were generated by inserting OLLAS or mSIGN-R1.Cytosol-OLLAS (GenBank accession no. EF635499, SEQ ID NO: 38) sequences into the multi-cloning site of pEGFP-N1 vector (Clontech).
Characterization of mAbs.
SDS-PAGE and Western blot analysis: 293T cells were transfected with the expression vectors for recombinant fusion proteins, harvested at day 2, lysed in RIPA buffer (150 mM NaCl, mM Tris-HCl, pH 8.0, 1% Nonident P-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitor cocktails (Sigma-Aldrich), and stored at −20 ° C. Each lysed sample was mixed with an equal volume of 2× SDS PAGE sample buffer and boiled at 95° C. for 5 min. Then the samples were separated in 12 or 15% SDS-PAGE and transferred onto PVDF membranes, followed by incubation with antibodies. Antibody-reactive bands on the blots were visualized by incubation with peroxidase-labeled secondary Ab followed by treatment with ECL Plus™ reagents (GE Healthcare). The isotypes of mAb heavy and light chains were determined by the Western blot analysis using mAb supernatant as primary Ab and rat isotype specific HRP-conjugated Abs (Southern Biotech) as secondary Ab.
Immunofluorescence: Stably transfected CHO cells and lymph nodes sections were examined for immunofluorescence as described previously. (Kang et al, Int Immunol. (2):177-86 (2003); Kang et al, Proc Natl Acad Sci USA. 101(1):215-220(2004)). Cells cultured on slides were washed with PBS, fixed in acetone for 5 min, and subsequently washed and blocked with PBS with 3% BSA for 1 hr. Then, after incubation with primary Abs for 1 hr at room temperature, cells were washed and incubated with fluorochrome-labeled secondary Abs. For tissue staining, peripheral lymph nodes from C57BL/6 mice were collected and embedded in Tissue-Tek OCT® (optimal cutting temperature) compounds (Sakura Finetek USA, Torrance, Calif.) before freezing at −80° C. Frozen tissues were sectioned 10 μm in thickness on a microtome and fixed in cold acetone for 15 min. Sections were incubated with primary Abs at room temperature in a humidified chamber for 1 hr, washed and then incubated with fluorochrome-labeled secondary Abs. Sections were mounted in Aqua-Poly Mount (Polysciences, Warrington, Pa.) and were stored at 4° C. until microscopic examination. The images were acquired with a deconvolution microscopy (Olympus, Melville, N.Y.) or with a Zeiss LSM 510 system (Carl Zeiss MicroImaging, Thornwood, N.Y.) at the Rockefeller University Bio-Imaging Resource Center.
FACS analysis: After detaching with 1 mM EDTA in PBS for 10 min, CHO/mDEC205 cells were incubated with primary Abs for 15 min at 4° C. Cells were washed, detected by Alexa 647 conjugated secondary Abs for 15 min at 4° C., and analyzed with FACSCalibur flow cytometer (BD Biosciences) at the Rockefeller University Flow Cytometry Resource Center.
Sequence analysis: The sequences of OFL and OLLAS were analyzed by PSIPRED (Bryson et al., Nucleic Acids Res. 33(Web Server issue), W36. (2005); McGuffin et al., Bioinformatics 16(4):404-405 (2000)) using the PSIPRED Protein Structure Prediction Server, and the NCBI BLAST database were searched for the sequence similarities.
To identify the epitopes of newly generated rat IgG mAbs L2 and L5, the inventors first made different forms of recombinant mLangerin proteins with/without E. coli OmpF derived flexible linker (OFL) sequences (
Another newly generated mAb L5 was specifically reactive to all the recombinant proteins containing a FLAG sequence, but not mouse Langerin itself, similarly to anti-FLAG mAb M2 (
To compare the binding sensitivity to the FLAG epitope between mouse IgG mAb M2 and the new rat IgG mAb L5, the inventors performed Western blot analyses with different FLAG tagged proteins. First, the purified protein of N-terminal FLAG tagged mLangerin ECD was loaded in serial, two-fold dilutions (
As shown in
To confirm the use of the new OLLAS epitope as a tag to detect fusion proteins, the OLLAS sequence (SGFANELGPRLMGK, SEQ ID NO: 1) to the N-terminus, the C-terminus, and internal sites of recombinant EGFP proteins was fused (
To compare the binding sensitivity of anti-OLLAS tag mAb OLLA-2 with anti-FLAG tag mAbs M2 and L5, EGFP protein was fused with FLAG and OLLAS tag in tandem at the N-terminus (
Next, to compare the binding sensitivity of mAb OLLA-2 with anti-V5 mAb (Invitrogen catalog number R960), human IgG1 Fc protein was fused with V5 epitope at the N-terminus and with OLLAS epitope at the C-terminus (
To test whether the OLLAS epitope was suitable as a tag for engineered mAbs, the sequence was fused to the C-terminus of the heavy chain of murinized NLDC145 (mNLDC145;
To confirm the usage of OLLAS epitope as a tag for engineered mAbs, the culture supernatants from 293T cells transfected with mNLDC145 or mNLDC145.OLLAS expression vectors were used for FACS analysis. Stable CHO cells expressing mDEC205 (CHO/mDEC205 cells) were incubated with each supernatant and stained with mAb OLLA-2 followed PE-conjugated anti-rat IgG prior to flow cytometry (
For fluorescent immunohistochemistry, mouse lymph nodes were incubated with rat IgG control, rat IgG mAb NLDC145, or engineered mNLDC145.OLLAS respectively. Subsequently, all lymph nodes were further stained with mAb OLLA-2 and anti-rat IgG labeled with Alexa488 and anti-B220. These results showed that mAb OLLA-2 was specific and sensitive to the OLLAS epitope and that there was no non-specific, background signals in the tissue staining of lymph nodes. These findings confirm that the OLLAS epitope and mAb OLLA-2 is broadly useful in various immunodetection methods, including FACS analysis and fluorescent immunostaining.
As shown in
In order to compare directly the use of mNLDC145.OLLAS and mAb OLLA-2, two engineered mAbs with the OLLAS epitope tag were generated (
Then, the inventors examined the lymph node tissues stained with either engineered mAb mNLDC145.OLLAS.OVA or rat mAb NLDC145 followed by visualization with Alexa647 labeled OLLA-2 and Alexa647 labeled anti-rat IgG, respectively (
A secondary structure prediction program called PSIPRED (Bryson et al., 2005; McGuffin et al., 2000) was utilized to characterize possible conformations of the OFL and OLLAS sequences used in-between the fused proteins that were generated successfully in previous (Galustian et al., Int Immunol. 16(6):853-866 (2004)) and current studies. As shown in
In a non limiting way, the instant invention may be summarized as follows.
Monoclonal antibodies against epitope tags are an efficient, convenient and rapid method for detecting recombinant protein expression (Jarvik and Telmer, 1998). If there is no antibody against the protein of interest, adding an epitope tag to this protein allows for protein detection with an antibody against the epitope sequence. For example, affinity tags such as a FLAG-tag appended to recombinant proteins have traditionally been used as a way of purifying proteins using standard conditions rather than developing individual biochemical purifications based on each protein's physical characteristics (Hopp et al., 1988).
Previously, rats were immunized to produce anti-mLangerin/CD207 antibody using two different forms of fusion proteins for mLangerin ECD expressed and purified from culture supernatants of stably transfected CHO cells (Cheong et al., 2007). As fusion partners of mLangerin ECD, FLAG epitope and flexible linker sequences from E. coli OmpF protein (OFL) were used. FLAG epitope was chosen for column purification and OFL was chosen to facilitate the folding of fusion proteins and to increase secretion from the cells. The OFL sequence as a linker was initially employed because the OFL sequence was viewed as highly flexible based on the molecular dynamics simulation of OmpF from E. coli (Im and Roux, J Mol Biol. 319(5):1177-1197 (2002)). Although the OFL sequence forms a beta-hairpin loop in OmpF, the secondary prediction by PSIPRED (Bryson et al., 2005; McGuffin et al., 2000) indicates that the OFL exists as a flexible coil in-between the fused proteins. This prediction is corroborated by the successful expressions of fusion proteins for C-type lectins, which were functional in sugar binding activities (Galustian et al., 2004) as well as immunogenic in mAb productions (Kang et al., 2004; Cheong et al., 2007).
In the process, it was found that new mAbs L5 and OLLA-2 were specific to the FLAG/OFL tagged mLangerin ECD protein, not to mLangerin ECD. Applicants demonstrate that L5 is a new anti-FLAG mAb (
MAb L2 or OLLA-2 is a novel rat IgG mAb against the newly identified epitope (SGFANELGPRLMGK, SEQ ID NO: 1) named OLLAS, with many advantages in recombinant protein engineering and immunodetection. First, this novel pair of tag and anti-tag mAb has a remarkable sensitivity and thus enhances the performance of a variety of immunodetection methods, including Western blot, immunocytochemistry, immunohistochemistry, flow cytometry, and immunoprecipitation. Especially, mAb OLLA-2 can detect the OLLAS epitope tagged proteins by Western blotting with more than 100 fold higher sensitivity than anti-FLAG M2 and anti-V5 mAbs. Second, the new OLLAS epitope can be fused to the C-terminal, the N-terminal, and the internal (or linker) sites of recombinant and fusion proteins including the engineered mAbs. The OLLAS epitope does not interfere with the biological activities of the recombinant proteins where it was inserted, such as fluorescence of EGFP and binding of antibodies, and can improve the expression of recombinant proteins as an inter-molecular linker between two recombinant proteins, as shown in the engineering of anti-mDEC205 mAb fused with ovalbumin. Third, mAb OLLA-2, as a rat IgG, can be used for the immunostaining of mouse and human samples without direct labelling of fluorochrome or enzyme. Because anti-tag mAbs and Abs are mostly made from mouse and rabbit, the anti-OLLAS tag mAb OLLA-2 from rat IgG will provide an extra advantage in immunodetection.
As indicated in
Thus, the present invention provides that mAb L5 is a new anti-FLAG with higher sensitivity, and that the novel OLLAS epitope and mAb OLLA-2 are superior tag and anti-tag mAb in terms of highly sensitive detection, broader range of applications, sequence uniqueness, and potential use as a linker. These new epitope tag and anti-tag mAbs will improve the current immunodetection methods for recombinant proteins.
All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.
This application is a Divisional of U.S. patent application Ser. No. 13/076,678 filed on Mar. 31, 2011, which application is a Divisional of U.S. patent application Ser. No. 11/974,384 filed on Oct. 12, 2007, the disclosures of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13076678 | Mar 2011 | US |
| Child | 13302136 | US | |
| Parent | 11974384 | Oct 2007 | US |
| Child | 13076678 | US |
