Patent Application
20110251106
The use of combinatorial approaches for protein identification, characterization and modification has been highly successful in both academic and commercial research and development. In this respect, filamentous bacteriophage, or phage, display technology has paved the way being the first library platform and still thrones as the dominating technology. Thus, phage display is widely applied in both basic and applied protein discovery, as well as in development of both novel protein-based diagnostics and therapeutic, which are the class of compounds most rapidly growing world-wide.
The principle of combinatorial phage display technology is based on the genotype—phenotype linkage offered by the property that each virion will only display on its surface the very same proteins that are encoded by the genome encapsulated by its protein coat. The phage particle itself is highly resistant to a variety of physiochemical conditions; hence phage display offers superior versatility in many selection regimes as compared to competing combinatorial technologies.
Phage display of heterologous polypeptides has been achieved using all five structural proteins of the filamentous phage coat, but only pIII- and to some extent pVIII-display have gained widespread use (
When the heterologous fusion is only a short peptide, multivalent display systems using phage genome-based vectors are preferred, whereas for larger fusions requiring folded domains most applications will benefit from the phagemid systems. In the latter case, antibody-pIII phage display is by far dominating the field, but alternative scaffolds are emerging at dawns early light, continuing the need for expansion of protein engineering tools of tomorrow. For many applications, it would be highly advantageous to be able to make, specifically and in a controlled manner, bispecific phage particles in that more than one of the coat proteins displayed a fusion peptide in the context of the same virus particle. Also, such a system should not interfere with already established display approaches and in particular pIII and pVIII display.
Endemann and Model, 1995 (PMID: 7616570), reported that the minor coat protein pVII was not accessible in the intact phage and that pVII was not functional with another protein fused to its N-terminus. Thus, this report concluded that pVII cannot be used for phage display.
Gao et al, 1999 (PMID: 10339535) and patent application WO0071694, describes heterologous peptide phage display on pVII using the octapeptide FLAG tag, as well as simultaneous phage display on pVII and pIX to generate functional heterodimeric polypeptides harbouring complex folding topologies (antibody Fv). These authors aimed at developing an alternative means for antibody display. The pVII and pIX fusion proteins were expressed from a phagemid employing a dicistronic constellation, hence the resulting functional phage particles inevitably contained varying amounts of pVII and pIX fusion proteins due to complementation by wild type pVII and pIX protein donated from the helper phage genome. As mentioned above, it had previously been suggested that pVII and pIX were not functional with another protein fused to their N termini, and Gao et. al. gave two possible reasons for their success, either alone or by the combination of both.
One possible reason was that a prokaryotic leader sequence (signal sequence) was attached N-terminally to the fusion proteins, thus ensuring targeting of the recombinant protein to the periplasmic space and thereby prevented accumulation in the cytoplasm. Another possible reason was that the recombinant proteins were expressed from a phagemid, not a phage genome as by Endemann and Model, hence wild type pVII and pIX from the helper phage inevitably needed for phagemid rescue were complementing the recombinant pVII and pIX fusion proteins, thus preserving wildtype functionality that otherwise may have been lost due to the recombinant modification. I.e. the phages would comprise a mix of wild-type and fusion proteins. The authors mention that the pVII-pIX display format would be particular useful for combinatorial display of heterodimeric arrays, which, for unknown reasons, appear to yield a particular powerful enrichment during panning protocols. The authors do not envisage using pVII as sole displaying protein (as phagemid or phage genome) or using pVII display in combination with display at another coat protein (different from pIX) to achieve bispecific display.
Kwasnikowski et al. (PMID: 16277988) described genetically stable fusion of scFv fragments to gene VII directly in the phage genome. I.e. the resulting phages comprised no native pVII protein, and the pVII display was multivalent. The authors speculate that one of the reasons for successful pVII display in the phage genome format is that they supported the fusion gene with a prokaryotic signal sequence that directs the fusion protein to the periplasmic space. The authors argued that the unique feature of their system is that the pVII displaying phages bears unmodified, wild-type pIII minor coat protein. Since it has been reported that multiple copies of functional pIII are required for host cell infection, the presence of wild-type pIII of the phage surface may facilitate recovery of selected antibodies with larger diversity. Thus, the authors do not envisage bispecific display, nor do they envisage pVII display without a prokaryotic signal sequence targeting to the periplasmic space.
Khalil et al (PMID: 17360403) describes an application exploiting the feature of a bispecific filamentous phage virion in which an exogenous peptide is displayed at each distal tip of the very same virion. They achieved this by using the combination of a common pIII phage genome vector complementing a pIX display phagemid. In this setting, the phage genome vector served as a helper phage in rescuing the phagemid, thus being reminiscent of the approach described herein of creating a bispecific phagemid virion by rescuing a pIII display phagemid by the use of a pVII modified helper phage genome. Moreover, the bispecific virions of Khalil et al display a peptide-pIII fusion that allows for a controlled biotinylation of their virion. There are however, several features that differ between these two avenues of obtaining a bispecific virion, as well as obtaining defined virion biotinylation, which make them unique from each other.
Firstly, the approach of Khalil et al cannot be used in combination with pIII phagemid display, as it is their phage genome vector that carries their pIII fusion, hence bispecificity cannot be obtained upon phagemid rescue and it would also highly likely be deleterious to the functionality of both pIII fusions.
Secondly, and as the authors also themselves pinpoint, genomic pIX modifications are not regarded as a viable strategy due to overlapping genes in the phage genome, thus they do not envision or speculate in making any modified helper phage genome that can be used for pIII phagemid (or pVIII) rescue and by this way donate a defined phenotypic feature to both distal tips of the very same virion. Khalil et al do never mention the use of modified pVII in either phagemid, or phage genome display.
Thirdly, Khalil et al do not speculate in modifying a single phage genome to achieve a bispesific virion, by exploiting simultaneous modification of more than one capsid gene within the very same genome. They merely use standard pIII peptide display through a commercially available phage genome vector.
Forth, Khalil et al only make bispecific virions displaying short peptides, not folded domains, and do never speculate in exploiting such display at either on, or both modified capsid proteins.
Fifth, Khalil et al achieve site-specific biotinylation of their pIII displayed peptide through in vitro chemical conjugation, not by an enzymatic reaction either in vitro or in vivo. The authors never envision enzyme mediated biotinylation of a displayed moiety by displaying an enzymatic substrate such as AviTag.
Finally, does Khalil et al. not show any type of display without the use of a N-terminal signal sequence.
An object of the present invention is to provide an alternative scaffold for peptides displayed on filamentous phages.
A first aspect of the invention is a pVII fusion proteins originating from a filamentous phage, said fusion proteins does not comprise an N-terminal signal sequence and thus is a direct fusion to an exogenous peptide.
Another aspect of the invention relates to nucleic acids encoding the fusion proteins of the invention.
One aspect of the invention relates to filamentous phages comprising the fusion proteins of the invention.
Another aspect of the invention relates to a library of filamentous phages.
One aspect of the invention relates to a phage display system comprising a phagemid and a helper phage, wherein the helper phage comprises a nucleic acid encoding the pVII fusion proteins of the invention.
Another aspect of the invention relates to a phage display system comprising a phagemid and a helper phage, wherein the phagemid comprises a nucleic acid encoding the pVII fusion proteins of the invention.
One aspect relates to a kit comprising a phage display system comprising a phagemid and a helper phage, wherein the helper phage comprises a nucleic acid encoding the pVII fusion proteins of the invention.
Schematic drawing of the filamentous phage structure. The virion is built up by five structural proteins that coat a single-stranded DNA molecule. In the wild type (wt) phage there are about 2700 copies of pVIII and approximately 3-5 copies of either of the four proteins pIII, pVI, pVII and pIX, which are found at each tip of the virion. Virion size is dependent on the genome size at approx. 2.3 nucleotides per pVIII coat protein and thus the length of the particle is accommodated by an increase or decrease in the inserted copies of pVIII. Notably, the pIII and pVIII structures have been characterized by x-ray fiber diffraction, crystallography and NMR. The minor coat protein pIII contains three distinct domains separated by glycin-rich regions: N1 (binds to TolA), N2 (binds to the F pilus) and CT (integrated into the virion and is important for normal virion assembly).
E. coli K12 codon optimisation of AviTag™, HIS6-tag and FLAG-tag. (A) Comparison of the commercially available AviTag™ DNA sequence with the E. coli K12 codon usage. Red columns are the submitted sequence and black columns are the reference set. (B) Upper line shows the original AviTag™, whereas the lower line shows the modified sequence adjusted according to the result in A. (C) codon optimised FLAG peptide. (D) codon optimised HIS6 peptide.
Titer of modified helperphages compared to wt helperphage.
ELISA analysis M13K07 AviTag-pVII
Normalised phage preparations were mixed with Streptavidin (SA) beads to absorb biotinylated virions and ELISA was performed as described in example 1.
ELISA analysis showing the accessibility of the FLAG-tag as a pVII fusion in M13K07. Normalised phage preparations were used in the ELISA assay. There is a specific FLAG-tag detection only of the M13K07-FLAG both for the M2 and M5 MAb. There is a stronger detection of the FLAG-tag by the M5 MAb.
Analysis showing the accessibility of HIS-tag as a pVII fusion to both M13K07 (SEQ ID NO: 31) and VCSM13 (SEQ ID NO: 32). Normalised phage preparations were mixed with Talon Dynabeads to absorb HIS6-tagged virions and ELISA was performed as described in example 1.
(A) Phagemid titers of scTcR and scFv-pIII displayed phagemids shown as cfuampR/ml. (B). Phagemid to helper phage ratios shown as the ratio of the phagemid titer (cfuampR/ml) divided by helper phage titer (cfukanR/ml).
ELISA analysis of scTCR phagemid AviTag showing specific accessibility of AviTag after phage rescue by streptavidin coated dynabeads. Inset show signal value of M13K07-AviTag helperphage. Normalised phage preparations were used.
ELISA analysis showing the accessibility of the FLAG-tag as a pVII fusion in two different phagemids, pFKPDNscTCR Vαβ4B2A1 (A) and pSEX-scFv anti-phOx (B) by capturing of phagemid virions by two anti FLAG antibodies, M2 and M5. Normalised phage preaparations were used.
ELISA analysis showing functionality of scTCRpIII (A) and scFvpIII (B) displayed on phagemid-derived virions with pVIIAviTag. Normalised phage preparations were used.
Analysis showing functionality of scTCR (A) and scFv (B) displayed on phagemid-derived virions with FLAG-tag and HIS6-tag. Normalised phage preparations were used.
Titre of genomic phage fUSE5-scTCRpIII with and without pVIIAviTag.
ELISA analysis showing the functionality of genomic fUSE5-AviTag phage preparations by capturing phages by streptavidin beads followed by detection of bound phages by anti M13-Antibodies. Normalised phage preparations were used.
ELISA analysis showing the functionality of pIII-displayed scTCR on genomic phage fUSE5 with Avitag-pVII. Normalised phage preparations were used.
Schematic drawing of the novel pGALD7 (A) and pGALD7ΔL (B) pVII display phagemids. The vector backbone of both phagemids was based on the pSEX81 (SEQ ID NO:29), which sequence can be accessed from GenBank accession no.: Y14584, and details on the constructed are described in Material and Methods. Both phagemids can accommodate cassettes of in frame exogenous sequences (termed E1 and E2) through easy cassette exchange of the NcoI/HindIII and MluI/NotI portions respectively. The cassettes are connected by a synthetic linker sequence that vary among the different constructs described herein.
Abbreviations: lacPO, lac promoter; sd, Shine-Dalgarno sequence; pelB, signal sequence of bacterial pectate lyase; TP, trypsine protease site; t, T7 transcriptional terminator.
Phagemid titers of scFv anti-phOx (SEQ ID NO:26) displayed from pGALD7ΔL (pVIIΔL), pGALD7 (pVII), pSEX81 (pIII) and pSEX81ΔL (pIIIΔL). All the phagemids harbour an ampicillin resistance marker, hence the titers are shown as ampicillin resistant colony forming units per millilitre solution (cfuampR/ml).
Antigen specific (phOx-BSA) ELISA comparing functional scFv anti-phOx (SEQ ID NO:26) display between pVII and pIII, and with and without signal sequence (ΔL). The ELISA was conducted as described in materials and methods and the titer input was 2×1010 cfuampR/ml for all samples, except for the pGALD7 (pVII), which was used undiluted (corresponding to 1.1×107 cfuampR/ml). The anti-M13HRP is a negative control on unspecific adsorbsion of the virion detection MAb to the antigen and block.
(A) Phagemid titers of scFv anti-phOx displayed from pGALD7ΔL (pVIIΔL), pGALD7 (pVII), pSEX81 (pIII) and pSEX81ΔL (pIIIΔL) shown as cfuampR/ml. (B). Phagemid to helper phage ratios shown as the ratio of the phagemid titer (cfuampR/ml) divided by helper phage titer (cfukanR/ml). The virion packaging was done as standard phagemid rescue as described in materials and methods (−), or with a final concentration of 0.1 mM IPTG present after super infection in both A and B.
Antigen specific (phOx-BSA) ELISA comparing functional scFv anti-phOx pVII display with and without signal sequence (ΔL) and with and without IPTG induction (0.1 mM) of the pVII fusion expression. The ELISA was conducted as described in materials and methods and the titer input was 2×1010 cfuampR/ml for pGALD7ΔL (pVIIΔL), whereas the pGALD7 (pVII) was used undiluted (corresponding to 2.0×109 and 1.1×107 cfuampR/ml without and with IPTG, respectively). The anti-M13HRP is a negative control on unspecific adsorbsion of the virion detection MAb to the antigen and block.
Antigen specific ELISA comparing functional scTCR (A) and scFv-anti-NIP (B) pVII display with and without signal sequence (ΔL). The ELISA was conducted as described in materials and methods using equal volumes on undiluted cleared supernatant. The anti-M13HRP is a negative control on unspecific adsorbsion of the virion detection MAb to the antigen and block. In (A), the GB113 antibody clone-specific for the 4B2A1 T cell receptor (Bogen et al, PMID: 1700755) was used as surrogate antigen substituting for the cognate I-Ed/λ2315 ligand to the scTCR Vαβ4B2A1 (Løset et al, PMID: 17925331).
(A) Phagemid titers of the scTCR Vαβ4B2A1 and the scFv anti-NIP (SEQ ID NO: 27) displayed from pGALD7ΔL and pGALD7 using standard phagemid rescue as described in material and methods. (B) Phagemid-to-helper phage ratios of the same samples as in (A).
(A) Cell density of the respective E. coli cultures at the end of the virion packaging protocol measure as optical density (OD) at A600nm. Notably, all cultures were initiated an identical density of A600nm 0.025 and super infected with M13K07 at MOI5 when A600nm 0.1 was reached. Packaging was then allowed to proceed ON at 30° C. before end culture OD was measured.
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
We here present a novel concept, in which the structural coat protein pVII of the filamentous phage virion is genetically altered such that the modified version encodes an N-terminal sequence tag. Depending on which type of tag that is fused to pVII, the virions are given the property of specific tag detection, as well as flexible purification and immobilization avenues as an inherent property of the system. The approach is directly compatible with all existing pIII and pVIII display systems whether phage genome-based of phagemid vectors are applied, including new library generation on pVII. Our concept therefore offers an unprecedented expansion of the already high versatility of phage display technology.
The current report shows for the first time that the filamentous phage genome tolerates an N-terminal peptide modification, not harbouring a signal sequence, of pVII without interfering with viability and functionality of the phage. This was true for both the M13K07 (SEQ ID NO: 31), VCSM13 (SEQ ID NO: 32) and fUSE5 (SEQ ID NO: 30) genomes as well as phagemids and as the sequence and phenotypic conservation between the various phage strains are very high, This most likely applies to all filamentous phages.
One of the pVII fusions chosen was a prokaryotic codon optimized version of the AviTag, a peptide which is the most efficient BirA substrate reported to date. By combining this pVII peptide display with pIII display we show that bispecific virions are produced. This was true for the phage-genome based vector fUSE5 (SEQ ID NO: 30) and from phagemid-based display when rescued with a modified M13K07 helper phage. It is easily conceivable that this bispecific nature can be used in combination with pVIII display as well. Particularly in the case of the phagemid-derived virions, the endogenous biotinylation level was very low.
However, if high biotinylation levels are desirable, this can easily be achieved by in vitro biotinylation of these virion, as well as by the use of in vivo biotinylation through the use of the novel F-positive E. coli AVB100FmkII strain.
Hence, the current concept allows for the combination of avidin-biotin technology (and other capture systems) with both dominating phage display platforms (phage and phagemid) and display systems (pIII and pVIII). It allows a controlled, site-specific attachment of the biotin moiety to the phage particle without interfering with the pIII and/or pVIII fusion, hence ensuring preserved functionality. The system is directly compatible with existing platforms without further modifications, only rendering the choice of use or not.
In conclusion, both genome-derived and phagemid-derived virions can tolerate the pVII modification, yielding virions with seemingly normal functionality and viability.
pVII Fusion Protein
In one aspect, the present invention provides a pVII fusion protein originating from a filamentous phage, said fusion protein comprising a fusion of an exogenous peptide to the N-terminus of pVII. Such a fusion protein is useful e.g. in the context of phage display.
When referring to an exogenous peptide, what is meant is a peptide not originally part of pIII, pVII or pVIII protein with or without any linker amino acids to the N-terminal end of the pIII, pVII or pVIII amino acid part of the fusion protein. In a preferred embodiment, the fusion protein does not comprise an N-terminal signal sequence. As used herein, the term peptide encompasses both short peptides, polypeptides, proteins and fragments thereof.
The term pIII protein refers to the amino acid sequence disclosed in SEQ ID NO 2. In one embodiment the pIII protein comprises the amino acid sequence with a sequence identity of at least 80% to that of SEQ ID NO 2, such as 80% identity, 81% identity, 82% identity, 83% identity, 84% identity, 85% identity, 86% identity, 87% identity, 88% identity, 89% identity, 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, or 99% identity.
The term pVIII fusion protein refers to a pVIII protein, or fragments thereof, fused to an exogenous peptide.
The term pVIII protein refers to the amino acid sequence in SEQ ID NO 3.
In an embodiment the pVIII protein comprises the amino acid sequence with a sequence identity of at least 80% to that of SEQ ID NO 3, such as 80% identity, 81% identity, 82% identity, 83% identity, 84% identity, 85% identity, 86% identity, 87% identity, 88% identity, 89% identity, such as 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, or 99% identity.
The term pVII protein refers to the amino acid sequence in SEQ ID NO 1.
In an embodiment the pVII protein comprises the amino acid with a sequence identity of at least 80% to that of SEQ ID NO 1, such as 80% identity, 81% identity, 82% identity, 83% identity, 84% identity, 85% identity, 86% identity, 87% identity, 88% identity, 89% identity, 90% identity, 91% identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, or 99% identity.
As commonly defined “identity” is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
Thus, in the present context “sequence identity” is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)×100). In one embodiment the two sequences are the same length.
One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilised. Alternatively, PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules. When utilising the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. “scoring matrix” and “gap penalty” may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.
The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
In a preferred embodiment, the term peptide refers exclusively to folded proteins such as antibody derived domains. The skilled addressee would recognise folded proteins could be antibodies or fragments thereof, covering Fv, scFv, Fab, single domains, Z domain of protein A (Affibody), Ankyrin or fragments thereof, T cell receptor or fragment thereof, MHC class I and II, Fibronectin or fragment thereof, Avimers, Anticalins, PDZ-domains, IgNAR or fragment thereof, CTLA4 or fragment thereof, ImmE7, Knottins, GFP and other gene-encoded biological fluorophores.
In principle, one can make a library of anything as long as it is displayed, hence at the highest level one can only separate between something that has only a non-structured configuration, as compared to a ordered structure, that is a fold.
In another preferred embodiment, the term peptide refers exclusively to short peptides between 2 to 50 aa. At some length a short random coil peptide will be long enough to adopt a defined secondary or tertiary fold and hence enter the folded domain definition. Obviously this will depend on chemical composition, hence one peptide of 20 aa will still be random coil, whereas another 20 aa peptide could be folded and hence fall into the folded domain definition.
In another preferred embodiment, the pVII fusion protein of the invention comprises a sequence selected from the group consisting of pos. 1-33, 2-33, 3-33, 4-33 and 5-33 of SEQ ID NO:1.
SEQ ID NO:1 (MEQVADFDTIYQAMIQISVVLCFALGIIAGGQR) is the amino acid sequence of structural coat protein pVII of the filamentous phage (wild type pVII). Most preferably, the pVII fusion protein comprises positions 1-33 of SEQ ID NO:1.
Preferably, the exogenous peptide is fused directly with or without any linker amino acids to the N-terminal end of the pVII sequence of the fusion protein. In yet another preferred embodiment, the pVII fusion protein does not comprise an N-terminal leader sequence.
The term “leader sequence” is used interchangeably with the terms “signal peptide” and “signal sequence”, and refers to an amino acid sequence that targets the protein (of which the leader sequence is part) to the periplasmatic membranespace of gram negative bacteria. Examples of leader sequences often used are pelBss, OmpAss, TorAss, malEss, phoAss, lamBss, Blass, and DspAss, mglBss, sfmCss, tolBss and TorTss. Such signal sequences are known to target the complete protein to the secretory machinery of E. coli which is known to include at least SRP-dependent, SEC-dependent, TatABC-dependent or YidC-dependent translocation from the cytosol to the periplasmic space (Baneyx et al. PMID: 15529165). Hence, the term N-terminal signal sequence refers to a signal sequence that is in the N-terminal part of the protein.
Signal sequences harbouring the property of targeting a protein (of which it is part) to the secretory machinery of E. coli and thereby translocate it from the cytosolic to the periplasmic compartment can be partly identified through signatures, or motifs, defined by the chemical property of their amino acid composition.
The variety of functional signal sequence existing is as of yet, however, exceeding the current knowledge in identifying them, hence current state of the art in defining a peptide as a cognate signal sequences are typically done through data mining using knowledge based data based as template by e.g. neural network or heuristic methodology. There are several such tools available to the community through open access channels as of today, such as SignalP, PPSEARCH of PROSITE (EMBL-EBI), SecretomeP, TatP.
The challenge is even higher with the class of secretory proteins, in the sense that they are exported from the cytosolic compartment, that deviate from the rules such that no signal sequence motif can be identified, but through data mining one can also here define signal sequence features or get the probability of the secretory capacity of the eukaryotic protein in question. As of yet, no such tool exist for the prokaryotic taxa.
The only method currently available that irrevocably identified a peptide as a signal sequence is therefore by experimental means to validate the property of a peptide to establish whether or not it is a real signal sequence. It is also clear that engineering may be performed in such peptides such that the given amino acid positions in the signal sequence may be altered, yet retain its function as a signal peptide, either by native functionality, or by altered functionality, such as increased transport capacity. Also deletion or addition of amino acids may be employed. Such analysis and engineering have indeed been done with the Ff pVIII signal sequence, g8 pss targeting the Sec-pathway, and the TorAss targeting the Tat-pathway. Especially the results of Shen et al may serve as well-founded guide lines for engineering of functional, but altered mutants, of the pIII signal sequence and the bacterial pectate lyase signal sequence.
The functionality of a signal sequence may be further broken down into the two following properties:
Though the vast majority of signal sequences map to situation 1) given above, it is clear that these proteins may be easily engineered into situation 2). Therefore, any currently known signal sequences e.g. a mutant pelBss and other that originally belong to the situation 1), but are altered into situation 2), are still regarded as cognate signal sequences.
Moreover, it is conceivable to either alter a signal sequence of situation 1) into situation 2), or directly choose a signal sequence that map to situation 2) and then after translocation remove the signal sequence. This can be done either by endogenous proteases of the host and/or in the case of e.g. phage display, when the protein is fused to a capsid protein. One would then engineer into the proper region of the signal sequence, or the protein of which it is a part, an artificial protease site, such that a defined cleavage can be performed. On can here envision two different types of protease sites chosen:
Both situations will still be regarded as signal sequence-dependent phage display. PS Wild Type Complementation
Hitherto, it was believed that pVII fusions without signal sequence were non-functional with respect to sustaining production of phage particles (Endeman et al, 1995; Gao et al, 1999). Therefore, pVII fusion proteins with an exogenous peptide fused directly to its N-terminus had to be complemented by wt pVII protein either from a second gene on the phage genome or by donation from a helper phage.
The term wild type, sometimes written wildtype, wild-type or wt, is the typical form of an organism, strain, gene, or characteristic as it occurs in nature. Wild type refers to the most common phenotype in the natural population. Wild type also refers to the allele at each locus required to produce the wild-type phenotype. Wild type is the standard of reference for the genotype and phenotype. In biology it relates specifically to the difference between a naturally occurring organism, and one that has been deliberately mutated. Site-directed mutagenesis is a research technique that allows for the mutation of specific nucleotides in the gene sequence of a wildtype gene. Wildtype proteins are written as wt-(name of protein) e.g. a wildtype pVII protein is written wt pVII, wt-pVII or wildtype pVII.
The present inventors have discovered that such pVII fusion proteins are indeed functional and need not necessarily be complemented by wt pVII protein.
Thus, one aspect of the invention relates to pVII fusion proteins that are functional in a phage display without complementation by wt pVII protein.
Kwasnikowski et al. reported pVII fusion proteins that did not have to be complemented by wild type pVII protein. However, the pVII fusion proteins of Kwasnikowski et al., comprised a signal peptide at the N-terminal end of the exogenous peptide. Said signal peptide was assumed to be necessary to direct the N-terminal pVII fusion protein into the periplasmic space and prevent its accumulation in the cytoplasm.
The absence of a signal peptide at the N-terminal end of the pVII fusion protein has various advantages. Signal peptides are normally proteolytically removed and this processing is often not complete which generates different N-terminal ends of the processed protein when a collection of proteins are expressed, thus introducing a random heterogeneity in the system, that may affect functionality of the proteins still harbouring the leader peptide leading to unwanted errors in the processed protein. This is prevented when no signal peptide is present.
Moreover, when a library of peptides are displayed, some of the peptides may prevent or affect proteolysis, which in turn will affect activity of the displayed protein and thus functional library diversity. Yet another surprising advantage of not including a signal peptide is that viability and functionality of the phage is not affected, as opposed to when using a signal peptide. Kwasnikowski et al., reported a reduced titer for phages with the pVII fusion protein comprising a leader sequence (signal peptide) at the N termini.
In one embodiment, the exogenous peptide is an affinity tag that binds to a predetermined target. The affinity tag may e.g. bind to a predetermined antibody. Pairs of affinity tags and predetermined targets are well-known to the skilled person.
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing. Tags are attached to proteins for various purposes.
Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique.
A feature of using an unprocessed N-terminal FLAG tag is that it has its formyl-Met residue intact and hence allows for the Ca2+ dependent interaction with the anti-FLAG MAb M1. The virion can thus be bound (that is immobilized) on M1 and liberated merely be chelating the cation by e.g EDTA, hence offering a very mild elution no extreme pH that denatures the heterologous fusion(s). By using the M1 this also means that the system can be used with other FLAG fusions present (internal, processed N-terminal, or C-terminal) without interference as these are not recognized by M1, or by simply keeping the [Ca2+] low.
In a preferred embodiment, the exogenous peptide of the pVII fusion protein is selected from the group consisting of Avitag (SEQ ID NO:4), FLAG tag (SEQ ID NO:9), HIS tag (SEQ ID NO:12), HAT tag, HA tag, c-Myc tag, Strep tag, V5 tag, antibody or fragment thereof, T cell receptor or fragment thereof, MHC class I and II, Ankyrin, IgNAR or fragment thereof, fibronectin or fragment thereof, Z domain of protein A, CTLA4 or fragment thereof, ImmE7, GFP and other gene-encoded biological fluorophores.
SEQ ID NO:2 (MSGLNDIFEAQKIEWHE) is a substrate sequence of the E. coli enzyme BirA sequence that enables enzyme mediated site-specific coupling of a biotin moiety to the substrate sequence. Thus, the assets of phage display technology and avidin-biotin technology are combined. Any fusion library in which the library is not displayed on pVII may e.g. first be fractionated against a target for identification of high-affinity library members and then immobilized using biotin binding to avidin, or an avidin-like matrix by means of also including the pVII fusion on the virions. Alternatively, any fusion library in which the library is not on pVII may e.g. first be immobilized, either randomly or in a predefined array on an avidin or avidin-like matrix, in a controlled, directional manner followed by target screening such as in e.g. SEREX, by means of also including the pVII fusion on the virions. Similarly, any member of such a pIII or a pVIII fusion library may be detected, either in bulk or as single clones, before or after target interaction by use of any avidin- or avidin-like-reporter complex the term reporter herein describes e.g. enzyme, nucleic acid species or synthetic or biological fluorophore.
Essentially the same rational as outlined for the AviTag, but whereas the latter results in a close to irreversible immobilization, the HIS6 allows for mild elution using imidazole. The HIS tag is compatible with all available IMAC matrixes,
In another preferred embodiment, the exogenous peptide of the pVII fusion protein is a library member. A library as used in the present context refers to a collection of different peptides. The peptides may be folded domains or short peptides of e.g. 2-50 amino acids. Such libraries are of interest because they can be used to identify new ligands binding to a given target. There are several advantages of using pVII for displaying a library as compared to libraries displayed using pIII or pVIII. pVII display contain the same assets as pIII display with respect to directionality and valence, but will not affect infectivity, a phenomenon known to occur with pIII display, which introduced uncontrolled and unwanted heterogeneity into the system upon e.g. rescue after affinity selection. Moreover, pVII display may be achieved without the need of an N-terminal leader peptide, which are prerequisites for both pIII and pVIII display. Finally, any target immobilised species in pIII display normally requires disruption (normally by competitive, or high or low pH elution) of this target-phage bond. This is e.g. known to severely hamper retrieval of high-affinity, or stable binders in pIII display. As pIII required for infection is unaltered and readily available for alternative interactions in pVII display even after phage-target interaction, this completely eliminates the need for bond disruption, e.g. acidic elution, as immobilised phages retain full infectivity and hence may be retrieved simply by infection whilst bound to target.
A second aspect of the invention is a nucleic acid encoding the fusion protein of the invention. The nucleic acid may be comprised within a phage genome or within a phagemid.
The term nucleic acid refers to a macromolecule composed of chains of monomeric nucleotides. In biochemistry these molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In addition, the term nucleic acids include artificial nucleic acids such as peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
A phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous phage Ff and plasmids to produce a vector that can propagate as a plasmid, and also be packaged as single stranded DNA in viral particles. Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques (transformation, electroporation). However, infection of a bacterial host containing a phagemid with a ‘helper’ phage, for example VCSM13 or M13K07, provides the necessary viral components to enable single stranded DNA replication and packaging of the phagemid DNA into phage particles.
A third aspect of the invention is a filamentous phage comprising the fusion protein of the invention. The filamentous phage may comprise a phage genome or a phagemid.
Phage, often called bacteriophage, is here meant as a virus infecting, replicating and which is secreted from bacteria. A filamentous bacteriophage, or filamentous phage, is a phage with a single stranded DNA genome (ssDNA genome) which is packaged with phage coatproteins The secreted filamentous phage particle has phenotypically a filamentous structure.
The term filamentous phage as used herein encompasses both phage genome-derived virions and phagemid-derived virions.
In one embodiment, the filamentous phage does not comprise a gene encoding the fusion protein, as the fusion protein may have been donated by a helper phage.
The term helper phage refers to a virus which helps a separate and unrelated defective virus defined as e.g. a phagemid which in itself is not a phage genome neither a functional virus, but merely a plasmid containing one or several elements derived from a phage genome, to reproduce by infecting the same host cell that is already occupied by the defective virus and providing the proteins which the defective virus is missing and needs to form complete its life cyclevirions containing the phagemid.
In another embodiment, the filamentous phage does comprise a nucleic acid encoding the fusion protein of the invention. The filamentous phage may comprise a phage genome or a phagemid. Particular preferred is a phage that comprises a phage genome comprising the nucleic acid encoding the fusion protein of the invention.
In yet another embodiment, the filamentous phage of the invention further comprises a gene encoding wt pVII and/or wt pVII protein. I.e. the number of fusion proteins displayed by the filamentous phage may be adjusted by modulating the ratio of wt pVII to pVII fusion protein. Such a system may also be referred to as a 77 system or 7+7 system depending on whether the wt pVII protein is donated from a helper phage (7+7) or from a second gene on the phage genome (77).
In still another embodiment, the filamentous phage does not comprise wt pVII gene and/or wt pVII protein. I.e. the filamentous phage comprises only pVII fusion protein and no wt. pVII protein.
In a preferred embodiment, the filamentous phage further comprises a pIII fusion protein or a pVIII fusion protein. A library may e.g. be displayed at pIII or pVIII and the pVII fusion protein may be used for affinity purification, immobilization or detection using e.g. avidin or an avidin-like matrix. Preferably, the filamentous phage comprises pIX protein solely in wild type form.
A fourth aspect of the invention is a library of filamentous phages of the invention, said filamentous phages displaying exogenous peptides or proteins as fusions to pIII, pVII or pVIII.
A library is a collection of filamentous phages displaying peptides or proteins as part of one or more of the filamentous phage coatproteins. Such libraries can comprise two or more phages displaying different peptides or proteins. In a preferred embodiment, peptides are displayed simultaneously at pVII and either pIII or pVIII.
In another preferred embodiment, the exogenous peptide displayed at pVII is selected from the group consisting of Avitag (SEQ ID NO:4), FLAG tag (SEQ ID NO:9), HIS tag (SEQ ID NO:12), HAT tag, HA tag, c-Myc tag, Strep tag, V5 tag, antibody or fragment thereof, T cell receptor or fragment thereof, Ankyrin, IgNAR or fragment thereof, fibronectin or fragment thereof, MHC class I and II, Z domain of protein A, CTLA4 or fragment thereof, ImmE7, GFP and other biological gene-encoded fluorophores. In this embodiment, the peptides displayed at pIII or pVIII are preferably library members. In an alternative embodiment, the library members are displayed at pVII, while pIII or pVIII displays an exogenous peptide selected from the group consisting of an Avitag (SEQ ID NO:4), FLAG tag (SEQ ID NO:9), HIS tag (SEQ ID NO:12), HAT tag, HA tag, c-Myc tag, Strep tag, V5 tag, antibody or fragment thereof, T cell receptor or fragment thereof, MHC class I and II, Ankyrin, IgNAR or fragment thereof, fibronectin or fragment thereof, Z domain of protein A, CTLA4 or fragment thereof, ImmE7, GFP and other biological gene-encoded fluorophores.
A fifth aspect of the invention is a phage display system comprising a phagemid and a helper phage, wherein the helper phage comprises a nucleic acid encoding the pVII fusion protein of the invention.
Phage display system, phage display technique, phage display technology or simply phage display refers to a method for the discovery and study of protein-protein, protein-peptide, and protein-DNA interactions that utilizes bacteriophage to connect proteins with the genetic information that encodes them.
Displaying protein or displayed protein refers to a protein fused to a phage coatprotein that is accessible for detection or immobilisation by a ligand
A sixth aspect of the invention is a phage display system comprising a phagemid and a helper phage, wherein the phagemid comprises a nucleic acid encoding the pVII fusion protein of the invention.
A seventh aspect of the invention is a kit comprising a phage display system composed of a phagemid and a helper phage, wherein the phagemid comprises the nucleic acid encoding the pVII fusion protein of the invention. The kit should include a phagemid with a pVII encoding gene with a multiple cloning site N-terminally in the coding region and a helper phage (e.g. M13K07, VCSM13 or other). The kit should be supplemented with protocols for infection, expression, immobilisation, selection and detection of phage clones. The kits should also be accompanied with necessary recipes for buffers and media for performing the specific assays.
A kit is here referred to a collection of reagents for generating phage particles with a single or bispecific fusion proteins either as a phage display library or as single phage particle. A kit could include phagemids, helper phages, bacterial strains and protocol with recipes for reagents and assay description. A kit can be used for the development of research, diagnostic and therapeutic reagents.
An Eighth aspect of the invention is a kit comprising a phage genome-based phage display system, wherein the phage genome comprises a nucleic acid encoding the pVII fusion protein of the invention.
The kit should include a phage genome vector (M13K07, VCSM13, fUSE5 (SEQ ID NO: 30)) with a pVII encoding gene with a multiple cloning site N-terminally in the coding region. The kit should be supplemented with protocols for infection, expression, immobilisation, selection and detection of phage clones. The kits should also be accompanied with necessary recipes for buffers and media for performing the specific assays.
A ninth aspect of the invention is a kit comprising a helper phage for production of pIII fusion phagemid libraries or single pIII fusion phagemid clones with a tag as a pVII fusion. The kit should include a Helper phage (M13K07, VCSM13) with a pVII encoding gene with inserted sequence encoding a short peptide suitable for capture and/or detection purposes. The kit should be supplemented with protocols for infection, expression, immobilisation, selection and detection of phage clones. The kits should also be accompanied with necessary recipes for buffers and media for performing the specific assays.
A tenth aspect of the invention is a kit comprising a phage genome vector for generating a phage genome library for display of fusion proteins on both pIII and pVII. Such a kit should include a phage genome vector (Ff) with genes encoding both PIII and PVII with multiple cloning sites N-terminally in each of the coding regions. Alternatively the kit should contain a phage genome vector with inserted sequence N-terminally in pVII encoding a short peptide suitable for capture and/or detection and a multiple cloning site N-terminally in pIII. The kit should be supplemented with protocols for infection, expression, immobilisation, selection and detection of phage clones. The kits should also be accompanied with necessary recipes for buffers and media for performing the specific assays.
An eleventh aspect of the invention is a method comprising the steps of
The invention will now be described in further details in the following non-limiting examples.
Modified helper phages M13K07 (SEQ ID NO: 31) and VCSM13 (SEQ ID NO: 32), with FLAG-pVII, HIS6-pVII, and AviTag-pVII may show a very broad potential for expanding the use of phage display technology, but it is of crucial importance that the fusion peptides do not compromise the functionality of the helper phage, thus titration of the phages are an important verification parameter. In addition the peptides fused to pVII must be accessible for the subsequent detection and/or immobilisation. This example support the fact that both pVII-modified helper phages can harbor a variety of peptides for detection and/or immobilization purposes and that these fusion peptides do not affect the infectivity of the phages.
Whilst early results from Endemann and Model (PMID: 7616570) indicated that the filamentous phage (Ff) capsid protein pVII did not tolerate exogenous fusions, it has later been shown that both phagemid-based (Gao et al (PMID: 10339535) and phage genome-based (Kwasnikowski et al (PMID: 16277988) peptide and folded domain display may be allowed as N-terminal fusions to pVII. In both cased, it is emphasized that the key to success required periplasmic targeting of the fusion protein by adding a prokaryotic signal sequence, or leader peptide, to the extreme N-terminus of the fusion, thus targeting the fusion to the SEC pathway of the E. coli host.
Productive pVII display has previously only been shown in the context of N-terminal fusions encoded on a phagemid harboring a N-terminal leader peptide ensuring transport of the recombinant pVII to the periplasmic compartment (Endeman et al, 1995; Gao et al, 1999).
However, it is known that before incorporated into the virion, wt pVII is found as an integral membrane protein in the inner membrane of the gram negative E. coli host, having its N-terminus facing the periplasmic space. Moreover, as this membrane bound, mature wt pVII retains its amino-terminal formyl group (Simons et al, PMID; 6945579), it does not appear to be N-terminally processed by e.g. the periplasmic leader peptidases, as is the case with the vast majority of signal sequence-directed proteins found outside the cytosolic compartment (Baneyx and Mujacic, PMID: 15529165). As no apparent signal sequence-like motif can be identified in the pVII ORF, its mode of translocation from the cytosol to the periplasm remains elusive, but most likely does not involved the four major secretory machineries identified in E. coli, namely the SEC-, SRP- and Tat- and YidC pathways (Baneyx and Mujacic, PMID: 15529165; Samuelson et al, PMID: 10949305). The structure of filamentous phage virion is shown in
All media and buffers were prepared essentially as described in Sambrook et al (Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press)). The anti-M13-HRP antibody and the M2 and M5 antibodies were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden) and Sigma-Aldrich (Oslo, Norway), respectively. Restriction enzymes (RE) were purchased from New England Biolabs (Ipswich, Mass., USA) with the exception of DpnI, which was obtained from Stratagene (LaJolla, Calif., USA). DNA oligos were purchased from MWG Biotech AG (Ebersberg, Germany). Dynabeads MyOne™-Streptavidin magnetic beads and Talon™ Ni-NTA magnetic beades were both purchased from Invitrogen (Oslo, Norway). BSA and Tween 20 was purchased form Sigma-Aldrich (Oslo, Norway). Pfu Ultra DNA and Phusion DNA polymerases were purchased from Stratagene (LaJolla, Calif., USA) and Sigma-Aldrich (Oslo, Norway), resepectively. TMB soluble was from Chalbiochem.
The E. coli strains XL1-Blue was purchased from Stratagene (LaJolla, Calif., USA). M13K07 helper phage was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden) whereas VCSM13 (SEQ ID NO: 32) was purchased from Stratagene (LaJolla, Calif., USA).
The open reading frame (ORF) of the AviTag™ (N-MSGLNDIFEAQKIEWHE-C) was compared to the codon usage in E. coli K12 strains using the GCUA server (http://gcua.schoedl.de/seqoverall.html). A prokaryotic codon-optimized version of the AviTag™ peptide sequence (SEQ ID NO:4) was attached N-terminally to the pVII ORF by QuikChange™ in vitro mutagenesis according to the manufacturers' protocol (Stratagen, LaJolla, Calif., USA), using the primer pair BirA-pVII_frwd/BirA-pVII_rev (5′-CCGGCTAAGTAACATGTCCGGCCTGAACGATATCTTTGAAGCGCAGAAAATTGAATGGCA TGAAATGGAGCAGGTC-′3/5′-GACCTGCTCCATTTCATGCCATTCAATTTTCTGCGCTTCAAAGATATCGTTCAGGCCGGAC ATGTTACTTAGCCGG-3′) (SEQ ID NO:5 and SEQ ID NO:6, respectively). In the same manner as described above, E. coli K12 codon optimized versions of the FLAG-tag (N-DYKDDDDK-C) (SEQ ID NO:9) and the HIS6-tag (N-HHHHHH-C) (SEQ ID NO:12) were attached N-terminally to the pVII ORF using the primer pairs FLAG-pVII-frwd/FLAG-pVII-rev (5′-CCGGCTAAGTAACATGGACTACAAAGATGACGATGACAAAATGGAGCAGGTCG-3′/5′-CGACCTGCTCCATTTTGTCATCGTCATCTTTGTAGTCCATGTTACTTAGCCGG-3′) (SEQ ID NO:7 and SEQ ID NO:8, respectively) and HIS6-pVII-frwd/HIS6-pVII-rev (5′-CCGGCTAAGTAACATGCATCACCATCACCATCACATGGAGCAGGTCG-3′/5′-CGACCTGCTCCATGTGATGGTGATGGTGATGCATGTTACTTAGCCGG-3′) (SEQ ID NO:10 and SEQ ID NO:11, respectively), respectively. The various constructs were verified by DNA sequencing (in-house ABI lab DNA sequencing core facility, Dept. Molecular Biosciences, University of Oslo) in all cases. To ensure a clean vector background, a BsrGI/SnaBI RE fragment containing the modified pVII was moved into either the M13K07 wt or VCSM13 wt genome on compatible RE sites using standard techniques. The DNA constructs were introduced into the various E. coli hosts by electroporation. Primer design was based on a sequence alignment of the M13K07 (New England Biolabs sequence) (SEQ ID NO:31) and VCSM13 (GenBank accession no.: AY598820) (SEQ ID NO:32) sequences using ClustalW. The sequence of the modified AviTag™-, HIS6-, and FLAG-sequences are shown in
Phages were amplified from E. coli XL1-Blue transformed with the M13K07 (SEQ ID NO: 31), VCSM13 (SEQ ID NO: 32) constructs essentially as described (Scott and Smith, PMID: 1696028).
10 μl/Tube™-Streptavidin beads were transferred to fresh 1.5-ml tubes and 500 μl 2% BSA in PBS (w/v) was added. Likewise, 250 μl of cleared supernatant or the appropriate amount of phages were transferred to 1.5-ml tubes and supplemented with 250 μl of 2% BSA. The tubes were then incubated for 1 h at room temperature (RT) on a rotating wheel. Thereafter, the beads were washed 3× by first immobilizing the beads by using a Dynal tube magnet rack. The supernatant was discarded and 0.5 ml of PBS containing 0.05% Tween 20 (PBST) added to each tube. The tubes were taken out of the rack and briefly vortexed before re-entered into the rack. The supernatant was again cleared and the washing repeated twice. The tubes were removed from the rack and 250 μl of blocked phage and 250 μl PBST were added to each tube. The tubes were then incubated for 1.5 h/RT on a rotating wheel. The tubes were washed 3× in PBST as described above. 0.5 ml of PBST containing anti-M13 MAb-HRP (1:2000) was then added to each tube and the tubes were incubated for 1 h/RT on a rotating wheel. The tubes were washed 3× in PBST as described above. 0.5 ml of ABTS was then added to each tube and the tubes left on the bench for 30 min, before place in the magnet rack and 100 μl supernatant transferred to Maxisorp ELISA strips (Nunc, Roskilde, Denmark). The absorbance was then measured at A405nm using a TECAN ELISA reader apparatus.
M2 and M5 antibodies were absorbed to MaxiSorp™ microtiter plate wells (Nunc, Roskilde, Denmark) in concentrations from 2.5 to 5 μg/ml in PBS, pH 7.4 overnight at 4° C. The wells were blocked with 2% skim milk in PBS (w/v) for 1 h at RT, virion preparations where then added and allowed to react for 1 to 2 h at RT before captured virions were detected with anti-M13-HRP (1:5,000) for 1 h at RT. Between each step, the wells were washed 3× with PBST. The wells were developed with ABTS substrate and the absorbance read at A405nm after 30 min.
16 ml 2×YT were inoculated with a fresh XL1-Blue culture and incubated at 37° C./250-rpm to an A600nm 0.4-0.8. 10 μl of each diluted phage preparations were transferred to a 96-well microtiter plate. 190 μl of the XL1-Blue culture was transferred to each well with the phage diluents. The plate was incubated for 50 min/37° C. BA82/20 membrane was overlaid a LB-kan agar-dish, a volume of 3 μl/sample spotted on the membrane and the dish incubated at 37° C./ON. Colonies were counted (
The BirA enzyme is an acetyl-CoA-carboxylase and is found endogenously in all E. coli. It has indeed been shown that the introduction of AviTag in the context of phage into such cells results in a small level (˜7%) of target biotinylation by endogenous BirA (Sholle et al, PMID: 16628754). To test whether or not the N-terminal pVII modification actually were functional in that virions were assembled and worked as an enzymatic substrate for BirA, we tested if the resulting virions could be captured from crude supernatant using SA-coated magnetic beads.
Capture of M13K07-AviTag pVII by Dynal Streptavidin beads. Two phages were employed in the assay: M13K07-AviTag which was in vivo biotinylated by the endogenous BirA- from the host and M13K07 wt. The result clearly showed a specific SA capture, whereas the M13K07 (SEQ ID NO: 31) did not bind. Thus, the AviTag-pVII fusion must indeed be functional in the sense that it both accommodate to the virion as wt pVII, whereas the N-terminal AviTag is accessible to the BirA enzyme and is recognized as a substrate for biotinlylation (
ELISA assays were performed to show the accessibility of the FLAG-tag as a pVII fusion in M13K07 (SEQ ID NO: 31) by capturing of phages by two anti FLAG antibodies, M2 and M5. In the assay wildtype M13K07, M13K07-His and M13K07-AviTag were included (
Both M13k07-HIS6 and VCSM13-HIS6 were tested for specific binding to DynalTalon Beads (IMAC matrix).
Briefly, Talon Beads were blocked by incubation with 2% BSA for 30 minutes with rotation. The beads were washed and added 250 μl of titer matched BSA-blocked phage supernatant (corresponding to 2×1010 cfukanR/ml) and further incubated for 30 min/RT on a rotating wheel. After washing the beads in PBST, anti-M13 MAb-HRP (diluted 1:2000) was added to each tube and the tubes were further incubated for 45 min/RT on a rotating wheel. After washing, ABTS was added to each tube and incubated 15 min RT, before placing in the magnet rack. 100 μl volumes of each solution transferred to Maxisorp ELISA strips. The absorbance was measured at A405 nm using a TECAN ELISA reader apparatus. The result is truly indicative that the HIS6-pVII containing virions are preferentially bound to Ni-NTA magnetic beads. Despite the low signals, which can be overcome by assay optimalisation, there is indeed a differential binding of the cognate virions to the Ni-NTA matrix. Of the most attractive applications of this particular pVII fusion is the possibility to exploit it for Ni-NTA purification in combinations with e.g. spin columns, as well as site-specific, and thus homogenous directional immobilization the Ni-NTA matrixes (
The promise of the invention is the use the modified helper phages for functional packaging of phagemids displaying a folded domain on a phage coat protein other than pVII, preferably in pIII or pVIII. The following examples support that modified helperphages with different peptides fused to pVII are able to perform functional phagemid packaging and that these phagemids display both functional pVII peptide fusion as well as functional folded domains fused to their pIII coatproteins. In this manner the examples also serve for bispecific display using phagemids.
All media and buffers were prepared essentially as described in Sambrook et al (Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press)). The anti-M13-HRP antibody and the M2 and M5 antibodies were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden) and Sigma-Aldrich (Oslo, Norway), respectively, whereas the F23.2 and GB113 antibodies were a kind gift from Professor B. Bogen (Institute of Immunology, University of Oslo, Norway). Dynabeads MyOne™-Streptavidin magnetic beads were purchased from Invitrogen (Oslo, Norway). BSA and Tween 20 was purchased form Sigma-Aldrich (Oslo, Norway). The hapten 2-phenyloxazol-5-one (phOx) conjugated to BSA was prepared essentially as described elsewhere (Makela et al, PMID; 722243).
The E. coli strain XL1-Blue was purchased from Stratagene (LaJolla, Calif., USA). M13K07 helper phage was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). The pSEX81 (SEQ ID NO:29), phagemid harbouring a scFv with specificity against 2-phenyloxazol-5-one (phOx) coupled to bovine serum albumin (BSA) were kindly provided by Affitech AS (Oslo, Norway). The pFKPDN-scTCR Vαβ4B2A1 is described in (Loset et al 2007, PMID: 17925331) (SEQ ID NO:28).
Phagemid rescue from E. coli XL1-Blue using M13K07 helper phages and virion assembly was monitored by spot titration as described (Welschof et al, PMID: 9050877 and Koch et al, PMID: 11126120).
MAb M2, M5, F23.2, GB113, phOx-BSA were absorbed to MaxiSorp™ microtiter plate wells (Nunc, Roskilde, Denmark) in concentrations from 2.5 to 5 μg/ml in PBS, pH 7.4 overnight at 4° C. The wells were blocked with 2% skim milk, or 2% BSA in PBS (w/v) for 1 h at RT, virion preparations where then added and allowed to react for 1 to 2 h at RT before captured virions were detected with anti-M13-HRP (1:5,000), for 1 h at RT. Between each step, the wells were washed 3× with PBST. The wells were developed with ABTS substrate and the absorbance read at A405nm after 30 min.
Two phagemids with different folded domains were employed, pFKPDNscTCR Vαβ4B2A1 pSEX-scFv anti-phOx, displaying a scTcR and a scFv as pIII fusion, respectively. Both were packaged with three modified and wt M13K07 helperphages. Briefly, overnight cultures of the two phagemid clones were infected with modified and wt helperphages. After incubation, the culture were centrifuged and the bacterial pellet was resuspended in YT-medium with ampicillin and kanamycine and further incubated ON at 30° C. Cleared supernatants by centrifugation were used downstream. E. coli XL-1 Blue was infected with dilutions of phages and plated on ampicillin and kanamycin plates for the titration of phagemids and helperphages, respectively. (
Both packaged phagemids show high ratios, indicating successful and functional packaging by all three modified M13K07 helper phage formats.
B—Functionality of pVII Donated by Helper Phage in Phagemid pIII Display:
pVII-Avitag display: With respect to AviTag-pVII functionality, SA capture on beads was conducted essentially as described in example 1. Though the signals are low, there is indeed a specific differential capture of the AviTag-pVII containing virions (
pvII-Flag Display:
ELISA assays were performed to show the accessibility of the FLAG-tag as a pVII fusion in two different phagemids-derived virions, pFKPDNscTCR Vαβ4B2A1 (
FLAG-specific reactivity is obtained for the phagemid-derived virions packaged with the M13K07-FLAG, whereas all other samples are negative. I.e the packaged phagemid-derived virions display the FLAG tag as a functional pVII-fusion.
A. Functionality of pIII Phagemid Display.
Two different phagemid-derived virions, pFKPDNscTCR Vαβ4B2A1 and pSEX-scFv anti-phOx, both displaying AviTag (
ELISA assays were performed by capturing of phagemid virions by their specific targets, MAB GB113 which binds scTCR and phOx-BSA for the scFv anti-phOx (SEQ ID NO:26). BSA was used as block and phagemids rescued by wt M13K07 was used as a control. Briefly, targets were coated on ELISA plates ON at 4° C. The plates were washed and phagemid preparations were incubated on the plates for 2 hours at room temperature. The plates were washed and further incubated with anti-M13 HRP conjugated antibody. The signals was developed with ABTS and incubation at RT/30 min, followed by measuring the absorbance at A405 nm (
The results show that cognate Ag-reactivity is obtained for all packaged phagemids. This analysis thus confirms that the modified helper phages do not affect the pIII display, but merely donates a defined phenotype to the pVII protein on the very same virion.
The invention allows for the generation of a genomic phage vector with display properties on pVII coatproteins. Such display will not affect the infectivity of virions like pIII display. Furthermore, the invention fosters bispecific display on pVII and pIII/pVIII, or even all three coat proteins simultaneously. The following example supports bispecific display on pIII and pVII in a genomic phage display system showing that the construct behave completely like wildtype phages with respect to propagation, virion assembly, viron concentration, pIII display phenotype and that it indeed is selectively in vivo biotinylated at the pVII peptide fusion.
All media and buffers were prepared essentially as described in Sambrook et al (Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press)). The anti-M13-HRP antibody was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden) and the F23.2 and GB113 antibodies were a kind gift from Professor B. Bogen (Institute of Immunology, University of Oslo, Norway). Restriction enzymes (RE) were purchased from New England Biolabs (Ipswich, Mass., USA) with the exception of DpnI, which was obtained from Stratagene (LaJolla, Calif., USA). DNA oligos were purchased from MWG Biotech AG (Ebersberg, Germany). Dynabeads MyOne™-Streptavidin magnetic beads were purchased from Invitrogen (Oslo, Norway). dm5CTP was from Fermentas (Burlington, Canada) BSA and Tween 20 was purchased form Sigma-Aldrich (Oslo, Norway). Pfu Turbo DNA and Phusion DNA polymerases were purchased from Stratagene (LaJolla, Calif., USA) and Sigma-Aldrich (Oslo, Norway), respectively. The QIAquick PCR clean-up kit was from Qiagen (Qiagen, Hilden, Germany).
The E. coli strain XL1-Blue was purchased from Stratagene (LaJolla, Calif., USA), whereas the E. coli strains MC1061 and K91K were kind gifts form Dr G. P. Smith (Division of Biological Sciences, University of Missouri, USA) The pSEX81 (SEQ ID NO:29) phagemid harbouring a scFv with specificity against 2-phenyloxazol-5-one (phOx) coupled to bovine serum albumin (BSA) were kindly provided by Affitech AS (Oslo, Norway). The fUSE5-scTCR Vαβ4B2A1 pIII display vector is described in (Løset et al 2007, PMID: 17925331).
Design and In Vitro Mutagenesis of AviTag™-pVII
The open reading frame (ORF) of the AviTag™ (N-MSGLNDIFEAQKIEWHE-C) was compared to the codon usage in E. coli K12 strains using the GCUA server (http://gcua.schoedl.de/seqoverall.html). A prokaryotic codon-optimized version of the AviTag™ peptide sequence was attached N-terminally to the pVII ORF by QuikChange™ in vitro mutagenesis according to the manufacturers' protocol (Stratagen, LaJolla, Calif., USA), using the primer pair BirA-pVII_frwd/BirA-pVII_rev (5′-CCGGCTAAGTAACATGTCCGGCCTGAACGATATCTTTGAAGCGCAGAAAATTGAATGGCA TGAAATGGAGCAGGTC-'3/5′-GACCTGCTCCATTTCATGCCATTCAATTTTCTGCGCTTCAAAGATATCGTTCAGGCCGGAC ATGTTACTTAGCCGG-3′) (SEQ ID NO:5 and SEQ ID NO:6, respectively). To ensure a clean vector background, a BsrGI/SnaBI RE fragment containing the modified pVII was cloned into an unmodified fUSE5-scTCR Vαβ4B2A1 genome on compatible RE sites using standard techniques. The DNA construct was introduced into E. coli MC1061 by electroporation. Primer design was based on the published fUSE5 sequence (GenBank accession no.: AF218364) (SEQ ID NO: 30).
Construction of the Novel Genomic pVII Display Vectors pGVII and pGVIIΔL
Primer design and vector assembly was done essentially as described in the SeamLess protocol (Stratagene, LaJolla, Calif., USA). Using VCSM13 genome DNA (SEQ ID NO: 32) as template and the primer pair VCSM13_F/VCSM13_R (5′-ATCTCTTCCATGGAGCAGGTCGCGGATTTCGACACAATTTATCAGG-3′/5′-ATCTCTTCCATGTTACTTAGCCGGAACGAGGCGCAGAC-3′) (SEQ ID NO:19 and SEQ ID NO: 20, respectively), the complete genome was PCR amplified with Pfu Turbo polymerase essentially as described in the SeamLess protocol (Stratagene, LaJolla, Calif., USA). Likewise, pSEX81ΔL (the latter described in Example 4), and pSEX81 (SEQ ID NO:29) both harbouring a scFv anti-phOx (SEQ ID NO:26) unit, were used as template in a standard PCR using Phusion DNA polymerase (Sigma, Oslo, Normay) with the primer pairs pGALDL_F/pGAL_R (5′-TCTCTTCACATGGCCCAGGTGCAGCTGGTGCAG-3′/5′-ATCTCTTCCCATTCTGATATCTTTGGATCCAGCGGCCGCAC-3′) (SEQ ID NO: 22 and SEQ ID NO: 23, respectively). and pGAL_F/pGAL_R (5′-ATCTCTTCACATGAAATACCTATTGCCTACGGCAGCCGCTGGC-3′/5′-ATCTCTTCCCATTCTGATATCTTTGGATCCAGCGGCCGCAC-3′)) (SEQ ID NO: 21 and SEQ ID NO: 23, respectively), respectively, to amplify the scFv units. Following PCR, all three segments were purified by a PCR clean up kit (Qiagen, GmbH, Hilden, Germany) and RE digested with Earl. RE digested, gel purified segments were then ligated and electroporated into XL1-Blue using standard techniques. Colonies were expanded and verified for correct insert size by PCR screening in a standard PCR using the primer pair pVII_frwd/pVII_rev (5′-AGCAGCTTTGTTACGTTGATTTGG-3′/5′-GCAGCGAAAGACAGCATCG-3′)) (SEQ ID NO: 24 and SEQ ID NO: 25, respectively). The genomic pVII display vectors were denoted pGVII (having signal sequence-dependent scFv-pVII display) and pGVIIΔL (having scFv-pVII display without any signal sequence). These now contain the scFv ORF as an in-frame fusion N-terminal to pVII and preserving the correct positioning of its start codon to the upstream pV ORF important for normal transcription and translation. Notably, the assembly of these phage genome vectors can just as easily be made by a three step PCR assembly where the exogenous ORF is PCR amplified with 5′-primer tag overhangs complementary to the vector backbone, which can be spliced by PCR SOEing with complementary segments amplified from the 5′- and 3′-portion of the vector covering the site of insertion. An ideal portion of the phage genome should cover a segment including the two unique RE sites BsrGI/SnaBI that are found flanking the pVII ORF in all Ff genomes. RE digested SOEing product can then conveniently be inserted into a complementary RE digested vector backbone, as describe e.g in Example 1 and 3. Another convenient assembly avenue would be to make an artificial gene assembly of the appropriate fusion ORF completely by short overlapping oligonucleotides that may be annealed as one pot, ligated and PCR amplified by flanking primers. This strategy could render an identical fragment as in the SOEing approach, or be RE independent on which insertion into the phage genome could be based e.g. on recombination as described (Tillett and Neilan, PMID: 10481038). A blend of the techniques may also easily be envisioned.
fUSE5 phages were amplified from E. coli MC1061 essentially as described (Scott and Smith, PMID: 1696028). Virion assembly was monitored by spot titration as described (Scott and Smith, PMID: 1696028 and Koch et al, PMID: 11126120). Where applicable, virions were purified and concentrated by PEG/NaCl precipitation as described (Sambrook et al (Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press)).
F23.2, GB113 antibodies were absorbed to MaxiSorp™ microtiter plate wells (Nunc, Roskilde, Denmark) in concentrations from 2.5 to 5 μg/ml in PBS, pH 7.4 overnight at 4° C. The wells were blocked with 2% skim milk in PBS (w/v) for 1 h at RT, virion preparations where then added and allowed to react for 1 to 2 h at RT before captured virions were detected with anti-M13-HRP (1:5,000) for 1 h at RT. Between each step, the wells were washed 3× with PBST. The wells were developed with ABTS substrate and the absorbance read at A405nm after 30 min.
A—Titration of fUSE5-scTCR-Avitag Genomic Phages
Neither the wt, nor the pVII-modified fUSE5-scTCR Vαβ4B2A1 versions exhibit any host toxicity. There is no phenotypic difference between the pVII-modified fUSE5-scTCR Vαβ4B2A1 versions regarding virion production and PEG precipitation efficiency. Both the pVII-modified fUSE5-scTCR Vαβ4B2A1 versions yield close to maximum theoretical titers feasible with the fUSE5 system (
B—Functionality of fUSE5-VII-AviTag Fusion Peptide
This ELISA analysis is to test the functionality of genomic phage preparations by capturing virions by streptavidin beads followed by detection of bound phages by anti M13-Antibodies. Briefly, MyOne Streptavidin dynabeads were blocked with BSA, washed and incubated with titer-normalised samples of fUSE5 phages with (scTCR/pVII-AviTag) and without (scTCR/pVII) pVII-Avitag fusion peptides. Beads were washed and bound phages were detected with ani-M13-HRP conjugated antibodies. Signal was developed by addition of ABTS and measured at A405 nm using a TECAN ELISA reader apparatus. The results show that the pVII-BirA peptide is accessible and has been biotinylated, and thus serves as an immobilisation and detection tag for phage genome-derived virions (
C—Functionality of fUSE5− scTCR pIII-Fusion
This ELISA analysis is to test the pIII fusion functionality of phage genome-derived virion preparations with and without genome-encoded AviTag-pVII. ELISA assays were performed by capturing of phage virions by two different antibodies recognising the scTCR Vαβ4B2A1) (SEQ ID NO: 28); MAB GB113 and F23.2, respectively. Skimmed milk was used as negative control. Briefly, antibodies were coated on ELISA plates ON at 4° C. The plates were washed and phage titer normalised preparations were incubated on the plates for 2 hours at room temperature. The plates were washed and further incubated with anti-M13 HRP conjugated antibody followed by the addition of 100 μl ABTS and incubation at RT. The absorbance was measured after 20 min at OD405 nm using a TECAN ELISA reader apparatus. The result shows that the scTCR phenotype is indistinguishable between the two fUSE5 versions. Hence, the pVII modification does not appear to affect the phenotype of the phage in any respect (
All media and buffers were prepared essentially as described in Sambrook et al (Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press)). The anti-M13-HRP antibody was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden) and the GB113 antibody was a kind gift from Professor B. Bogen (Institute of Immunology, University of Oslo, Norway). Restriction enzymes (RE) were purchased from New England Biolabs (Ipswich, Mass., USA) with the exception of DpnI, which was obtained from Stratagene (LaJolla, Calif., USA). DNA oligos were purchased from MWG Biotech AG (Ebersberg, Germany). BSA and Tween 20 was purchased form Sigma-Aldrich (Oslo, Norway). Pfu Turbo DNA polymerase was purchased from Stratagene (LaJolla, Calif., USA). The haptens 2-phenyloxazol-5-one phOx and 5-nitrophenacetyl (NIP) conjugated to BSA were prepared essentially as described elsewhere (Makela et al, PMID; 722243 and Michaelsen et al, PMID: 2125362). Isopropyl-beta-D-thiogalactopyranoside (IPTG) was purchased from Fermentas (Burlington, Canada).
The E. coli strain XL1-Blue was purchased from Stratagene (LaJolla, Calif., USA). M13K07 helper phage was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). The pSEX81 (SEQ ID NO:29) phagemid harbouring a scFv with specificity against 2-phenyloxazol-5-one (phOx) coupled to bovine serum albumin (BSA) was kindly provided by Affitech AS (Oslo, Norway). The pFKPDN-scTCR Vαβ4B2A1 pIII display phagemid has been described elsewhere (Løset et al 2007, PMID: 17925331). The prokaryotic expression vector pSG1 harbouring the scFv anti-NIP (SEQ ID NO: 27) (unpublished) is based on pHOG21 (Kiprianov et al, PMID: 9005945) and has been made in-house from the antibody variable genes derived from pLNOH2 and pLNOK (Norderhaug et al, PMID: 9202712).
Construction of the novel pVII display phagemid vectors pGALD7 and pGALD7ΔL As a starting template for the vector backbone, the pSEX81 (SEQ ID NO:29) phagemid described above was chosen (GenBank accession no.: Y14584). Firstly, to remove the prokaryotic pelB signal sequence (N-MKYLLPTAAAGLLLLAAQPAMA-C) (SEQ ID NO:33) encoding strech in this vector, a NcoI RE site was introduced in the extreme N-terminus by QuikChange™ in vitro mutagenesis using the primer pair a41g-frwd/a41g-rev (5′-AGAGGAGAAATTAACCATGGAATACCTATTGCCTACGGC-3′/5′-GCCGTAGGCAATAGGTATTCCATGGTTAATTTCTCCTCT-3′) (SEQ ID NO:13 and SEQ ID NO: 14, respectively), thereby changing the first nucleotide in the second codon of the pelB ORF from A to G. Following mutagenesis, the vector was NcoI digested, re-ligated and used as template in a second PCR retrieving the relevant part of the vector using the primer pair pHOG_EcoRI_frwd/scTCR_rev (5′-TAGCTCACTCATTAGGCACCC-3′/5′-TTTGGATCCAGCGGCCGC-3′) (SEQ ID NO:15 and SEQ ID NO: 16, respectively). This PCR fragment was then moved into the original pSEX81(SEQ ID NO:29) on the compatible EcoRI/HindIII RE sites using standard techniques and confirmed by DNA sequencing. This step completely removed the pelB signal sequence encoding portion, but preserved the start codon and its relative position towards the lacPO and Shine-Dalgarno sequence (SD) important for normal transcription and translation, as well as adding only one Ala residue before the exogenous sequence defined by the NcoI/NotI RE sites found in the original pSEX81 (SEQ ID NO:29). The new construct was denoted pSEX81ΔL. Secondly, the pVII encoding sequence was amplified from M13K07 using the 5′-end RE-tagged primer pair pVII_EcoRV/pVII_NheI (5′-ATATGATATCAGAATGGAGCAGGTCGCGGATTTCG-3′/5′-ATATGCTAGCTTATCATCTTTGACCCCCAGCGATTATACC-3′) (SEQ ID NO:17 and SEQ ID NO: 18, respectively). This PCR fragment was then moved into both the pSEX81 (SEQ ID NO:29), and pSEX81ΔL phagemids on the compatible RE sites, thereby exchanging the pIII encoding region in both and resulting in a N-terminal in-frame pVII fusion of the NcoI/NotI-defined cassette in the original pSEX81(SEQ ID NO:29). The new constructs were confirmed by DNA sequencing and denoted pGALD7 and pGALD7ΔL, respectively. To switch the scFv anti-phOx (SEQ ID NO:26) unit in the various phagemids described above, with the scTCR Vαβ4B2A1 and scFv anti-NIP (SEQ ID NO: 27) units from pFKPDN and pSG1, respectively, this was done as NcoI/NotI RE defined cassette exchange using standard techniques. All phagemids described herein were introduced into E. coli XL1-Blue by electroporation using standard techniques.
Phagemid rescue from E. coli XL1-Blue using M13K07 helper phages and virion assembly was monitored by spot titration as described (Welschof et al, PMID: 9050877 and Koch et al, PMID: 11126120).
MAb GB113, phOx-BSA or NIP-BSA were absorbed to MaxiSorp™ microtiter plate wells (Nunc, Roskilde, Denmark) in concentrations from 2.5 to 5 μg/ml in PBS, pH 7.4 overnight at 4° C. The wells were blocked with 2% skim milk, or 2% BSA in PBS (w/v) for 1 h at RT, virion preparations where then added and allowed to react for 1 to 2 h at RT before captured virions were detected with anti-M13-HRP (1:5,000) for 1 h at RT. Between each step, the wells were washed 3× with PBST. The wells were developed with ABTS substrate and the absorbance read at A405nm after 30 min.
Prompted by the results from the modified helper phages described above, pVII display of folded domains was also assessed. As both Gao et al and Kwasnikowski et al have shown that such display is allowed when used in combination with signal sequence-directed periplasmic targeting, we constructed two novel phagemids termed pGALD7 and pGALD7ΔL, allowing for N-terminal pVII display with and without such a signal sequence, respectively (
The initial constructs contained a scFv unit based on human antibody variable segments and being specific for the hapten conjucate phOx-BSA. As with the pVII modified helper phages described previously, pVII display of the scFv should not interefere with normal virion assembly. We therefore compared the performance of these scFv anti-phOx pVII display phagemids with and without signal sequence and also with standard pIII display with and without signal sequence, using standard phagemid rescue and titration as described in materials and methods. (
The titration result indeed showed that phagemid-containing virions were made in all cases. However, whereas the pVIIΔL phagemid yielded titers about 30-fold lower than standard pIII display, there was a gross 105-fold reduction in the signal sequence-directed pVII display. As wt complementation of pVII is present from the helper phage in this system, this finding was both surprising and important, because it shows that the signal sequence-directed pVII display (pVII) severely interferes with the virion assembly process, whereas this effect is only minor in the case with signal sequence-less pVII display (pVIIΔL). In comparison, the titer difference between pIII display with and without signal sequence, was only minor.
Based upon the titers determined above, we then assessed the functional scFv display on these virion samples in a phOx-BSA specific ELISA using titer normalized inputs, except for the pGALD7-derived sample that was used undiluted as the phagemid titer was very low. (
The result clearly showed functional scFv display from both the signal sequence-less pVII version and standard pIII, whereas the other samples were negative. That the signal sequence-directed pVII display yielded negative results was expected due to the 2000-fold less virion input. It is known that pIII is exported to the periplasm through the SEC-pathway, hence the signal sequence-less pIII version yields pIII fusions defect in perplasmic targeting, which is a prerequisite for virion incorporation. The virions from this sample, hence contains only helper phage derived pIII (loss of physical phenotype-genotype linkage), though the phagemid packaging efficiency is close to normal and results in normal titers (as seen in
The scFv-pVII/pIII expression cassette in all the phagemids employed here are controlled by the lac promoter and the virion packaging was done using the standard protocol without IPTG induction (Welschof et al, PMID: 9050877), Thus, it should be possible to increase the scFv display by forcing stronger expression using IPTG during packaging. Moreover, an important feature of phagemid phage display is the fact that functional display is dependent upon helper phage mediated rescue of the phagemid. Therefore, in contrast to phage genome-based display, there are two sources of ssDNA that can be packaged into the virion from a given cell—the phagemid, or the helper phage genome. Importantly, both types of virions will have access to the very same pool of capsid proteins, as they are produced and found within the very same host cell. To ensure the preservation of the physical genotype-phenotype linkage that forms the very basis for combinatorial phage display technology, it is therefore of the out most importance that the phagemid-to-helper phage ratio is in favour of the phagemid. In a new experiment, we prepared the same phagemid-derived samples as described above, but now also comparing virion assembly with and with out IPTG included during packaging. During titration, we this time also mapped the helper phage genome titers by means of the kanamycin resistance found on the helper phage genome.
The current titration result (
Based upon the phagemid titers shown in
The result did indeed show the functional scFv-pVII display was again achieved with the signal sequence-less pGALD7ΔL and the phOx-BSA reactivity increased significantly upon IPTG-forced pVII fusion expression. This increase in antigen reactivity most likely reflects an increased number of pVII fusion per virion as well as an increased number of virions harbouring a pVII fusion per see. The latter is likely as it is known that in standard pIII display only between 1 to 10% of the phagemid-containing virions actually contains a fusion (Bradbury and Marks, PMID: 15261570). On the other hand, the signal sequence-directed pVII display again showed no functional phOx-BSA binding. Based on
So far, we have clearly shown functional pVII phagemid-based display of the scFv anti-phOx (SEQ ID NO:26), unit and that this construct is comparable to pIII display of the same scFv, exhibiting only a minor reduction in phagemid titers and in antigen binding capacity.
The scFv anti-phOx (SEQ ID NO:26) has been selected for a human antibody scFv library and is known to express rather well in E. coli (Marks et al., PMID: 1748994). To see whether or not pVII display exhibits the capacity to functional display of more challenging fusion partners, we therefore subcloned a scFv anti-NIP (SEQ ID NO: 27) based on the antibody variable genes of a murine hybridoma, as well as a scTCR based on the variable genes from the murine T cell clone 4B2A1 (Løset et al, PMID: 17925331) into both pGALD7ΔL and pGALD7. It is well known that many hybridoma variable genes do not express well in E. coli and also when phage displayed (Krebber et al, PMID: 9032408) and T cell receptors are on class of folded proteins that has proven especially difficult to accommodate to phage display (Li et al, PMID: 15723046, and Løset et al, PMID: 17925331).
Virions from these new phagemids were prepared by standard phagemid rescue and tested for their antigen binding capacity in ELISA (
The result did indeed show that functional pVII display was achieved both with the scFv anti-NIP (SEQ ID NO: 27) and the scTCR Vαβ4B2A1 and in contrast to the what was observed previously with the scFv anti-phOx (SEQ ID NO:26), this was the case both with and without signal sequence-directed pVII display. The signals observed in
The phagemid titers again showed that the signal sequence-less pVII display (pGALD7ΔL) exhibited superior performance as compared with signal sequence-directed pVII display (
In light of the results above, it was noteworthy the see that there was a quite distinct difference between the scTCR and scFv anti-NIP with respect to host cell proliferation during virion packaging (
What is evident from
All media and buffers were prepared essentially as described in Sambrook et al (Molecular cloning: a laboratory manual (Cold Spring Harbor Laboratory Press)). The E. coli strains XL1-Blue and AVB100 (based on MC1061) were purchased from Stratagene (LaJolla, Calif., USA) and Avidity (Denver, Colo., USA), respectively.
To obtain F plasmid positive E. coli AVB100 (chromosomal StrR) the cells were mated with XL1-Blue (F plasmid TetR) as follows. Single colonies of each strain was inoculated into 5 ml LB medium supplemented with the appropriate antibiotic and incubated over night at 37° C. with rigorous shaking. The next day, fresh 5 ml cultures were initiated at an A600nm of 0.1 and grown to mid log phase at 37° C. with rigorous shaking before 1 ml of each were mixed and incubated stationary at 37° C. for 1 h. Thereafter, 10 μl of this mixture was transferred to 5 ml fresh LB medium containing 100 μg/ml Str and 30 μg/ml Tet and incubated over night at 37° C. with rigorous shaking. The next day, dilutions of this culture was spread on agar dishes containing 100 μg/ml Str and 30 μg/ml Tet and the resulting colonies used as source of the new F plasmid positive AVB100 strain, termed AVB100FmkII.
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2007 01673 | Nov 2007 | DK | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP08/60908 | 8/20/2008 | WO | 00 | 10/12/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60956871 | Aug 2007 | US |
