Patent Application
20170261498
The invention concerns a fusion polypeptide comprising several molecules of folding helper polypeptides, comprising one multimerization domain, in particular Skp, and at least one molecule of SlyD or SlpA, wherein no further target polypeptide sequences are fused to said fusion polypeptide. The invention further concerns an immunoassay and the use of said fusion polypeptide in an immunoassay for reduction of interferences or minimizing false positive results or for stabilizing proteinaceous assay reagents. Further the invention concerns a reagent kit for use in an immunoassay comprising said fusion polypeptide.
Chaperones, which are known as classical folding helpers, are proteins that assist the folding and maintenance of the structural integrity of other proteins. They bind to denatured or hydrophobic surfaces of proteins and help in re-naturing and keeping proteins in solution. Due to their superior physico-chemical properties chaperones are used as folding assistants and fusion partners in protein technology. One class of chaperones is the family of FKBP chaperones, proteins that bind to the immunosuppressant drug FK506.
The use of FKBP chaperones like SlyD, FkpA and SlpA (=SlyD-like protein A) as fusion partners for difficult proteins has been widely described (WO 2003/000878, WO 2009/074318, EP 2127679).
Commercially available immunoassays for the detection of antibodies against pathogens like, e.g., human immunodeficiency virus (HIV), Rubella virus, cytomegalovirus (CMV) or herpes simplex virus (HSV) contain polypeptide fusion proteins wherein chaperones are fused to specific target antigen sequences. Such fusion proteins are described in, e.g., Scholz et al., J. Mol. Biol. (2005) 345, 1229-1242, Scholz et al., Biochemistry (2006) 45, 20-33 or Scholz et al., Biochemistry (2008) 47, 4276-4287.
SlyD, FkpA and SlpA possess outstanding solubilization (i.e. chaperone) properties and are characterized in that they are able to refold reversibly after chemically or thermally induced unfolding. As fusion partners for difficult target polypeptides they play at least a threefold role: firstly, they increase the production of target proteins that are heterologously overexpressed in procaryotic organisms, secondly, they facilitate and assist the in vitro refolding of the target polypeptides, and thirdly, they increase the overall solubility and stability of the respective target polypeptide.
However, chaperones like SlyD, FkpA and SlpA are immunogens in their own right. Since they are abundant bacterial proteins, they are recognized as non-self by the human (or, generally speaking, mammalian) immune system, triggering a powerful humoral immune response, which results in the production of specific antibodies with high affinity. A considerable percentage of adult human sera therefore contain significant immunoglobulin titers against these chaperones. As a consequence, there is a considerable likelihood that a human serum sample may turn out false positive in an immunoassay, in particular in an immunoassay of the double antigen sandwich format that uses antigen specifiers fused to bacterial chaperone modules.
In order to avoid such unwanted cross-reactions due to the antibody-induced bridging of fusion partners, immunoassays are usually designed in an asymmetric fashion. This means that for example in an immunoassay for the detection of antibodies designed in the well-known double antigen sandwich format (DAGS) a person skilled in the art uses different fusion partners for the applied antigens on both sides of the assay in order to avoid non-specific bridging. If identical fusion partners were used for the antigens on the solid phase and the detection side, interfering components in the sample could establish a bridge between said identical fusion partners and thus evoke a (false) positive reaction.
As a further means to prevent unwanted binding to a fusion partner which is part of an antigen-fusion protein, a chemically polymerized form of the employed fusion module (i.e. the fusion part without any specific antigen) is usually added to the assay in large excess. Due to their high epitope density and their high effective concentrations, these chemically polymerized fusion modules preferably allure, bind and quench those IgGs and IgMs that are directed towards said fusion module. The chemically polymerized fusion modules serve as a bait, and they quench the interfering compounds in the sample very efficiently so that interferences can be suppressed and ruled out. When, for instance, E. coli SlyD is used as a fusion partner for a given antigen in an immunoassay of the double antigen sandwich type, it would be highly advisable to generate chemically polymerized E. coli SlyD (by means of cros slinking with, e.g., glutardialdehyde) and to add this polymer to the assay as an anti-interference substance.
However, a considerable disadvantage in using chemically polymerized proteins lies in the chemical production process itself. Depending on the cross-linking agent applied, the chemical polymerization process is not entirely reproducible. The chemically cross-linked polymers usually show a large distribution of polymers of different size, i.e. they strongly vary with respect to connectivity and they are characterized by considerable heterogeneities. In order to select the effective polymer fractions (i.e. the polymer fractions with the desired anti-interference abilities) the polymer pool needs to be purified and fractionated by time-consuming and cumbersome chromatographic methods. In addition, only limited yields can be obtained as only a small percentage of the product will elute in the desired fraction.
In order to overcome the obstacle of using insufficiently characterized chemically polymerized material in immunoassays we searched for an alternative way to generate anti-interference substances. We strived to obtain anti-interference modules with a sufficiently high and well-defined epitope density in a simple and convenient manner. So we addressed the question whether it was possible to create a well-defined, highly soluble and highly efficient anti-interference module in an utterly recombinant fashion. Briefly, the problem to be solved was to obtain chaperone fusion partners in a soluble form, with high epitope density and in a reproducible and standardizable way.
The problem is solved by the current invention as characterized by the claims. In particular, the invention concerns a fusion polypeptide comprising several molecules of folding helper polypeptides, comprising one multimerization domain and at least one molecule of SlyD or SlpA, wherein no further target polypeptide sequences are fused to said fusion polypeptide. As a preferred multimerization domain Skp is used. Preferably one molecule of Skp is fused to two adjacent molecules of SlyD or to two adjacent molecules of SlpA. In another preferred embodiment one molecule of Skp is N-terminally fused to two adjacent molecules of SlyD or to two adjacent molecules of SlpA or to another monomeric chaperone that serves as a fusion partner. The term “N-terminally fused” means that Skp is fused to the N-terminal end of another protein molecule, in this case to the N-terminal end of either SlyD or SlpA. In a further preferred mode the fusion polypeptide according to the invention comprises SEQ ID NO. 1 which can also be named Skp-tandem-SlyD or Skp-SlyD-SlyD. A preferred fusion polypeptide is a polypeptide consisting of SEQ ID NO. 1 (Skp-tandem-SlyD). A further preferred fusion polypeptide is a polypeptide comprising SEQ ID NO. 9 which can also be named Skp-tandem-SlpA or Skp-SlpA-SlpA. Particularly preferred is a polypeptide consisting of SEQ ID NO. 9.
Another embodiment of the invention is the use of a fusion polypeptide as an additive in an immunoassay or as an additive to an assay reagent so that the fusion polypeptide can be used for reduction of interferences or for minimizing false positive results. According to the invention said fusion polypeptide can also be used for increasing the solubility of proteinaceous ingredients within an assay reagent. Also encompassed by a further preferred embodiment of the invention is a reagent kit for the detection of an analyte in an isolated sample by an immunoassay which comprises said fusion polypeptide.
In a further preferred embodiment a method for detecting an analyte in an isolated sample is encompassed wherein a fusion polypeptide as characterized above is used as a reagent for reduction of interference or for minimizing false positive results.
Another embodiment of the invention is a method for detecting an analyte such as, e.g., an antibody in an isolated sample, said method comprising
a) forming an immunoreaction admixture by admixing a body fluid sample with a specific binding partner that can be specifically bound by said analyte present in said sample
b) adding a fusion polypeptide according to the invention to said immunoreaction admixture either before, at the same time or after said specific binding partner is added to said sample
c) maintaining said immunoreaction admixture for a time period sufficient for allowing the analyte present in said body fluid sample to immunoreact with said specific binding partner to form an immunoreaction product; and
d) detecting the presence and/or the concentration of any of said immunoreaction product.
A further aspect of the invention is a reagent kit for the detection of an analyte, in particular for the detection of an antibody, in an isolated sample by an immunoassay, said kit comprising a fusion polypeptide according to the invention. Other ingredients of a reagent kit are known to someone skilled in the art and include specific binding reagents such as e.g. antigens. Further kit components are buffers, preservatives, labeling substances and instructions for use.
SEQ ID NO. 1 shows the amino acid sequence of Skp-tandem-SlyD or Skp-SlyD-SlyD. The glycine-rich spacer region (underlined) between the Skp and SlyD units has been added to enable maximal flexibility of the fusion partners and to make sure that the Skp units may form ordered trimers without any interference of the C-terminally fused SlyD proteins. An additional C-terminal octa-histidine tag has been added for purification purposes (see experimental section). SEQ ID NO. 1 comprises amino acid residues 1-165 of E. coli SlyD (complete molecule cf. SEQ ID NO. 3) in tandem form, i.e. two E. coli SlyD (1-165) units in a row.
GGMKVAKDLV VSLAYQVRTE DGVLVDESPV SAPLDYLHGH
SEQ ID NO. 2 shows the complete amino acid sequence of E. coli Skp (161 aa) according to SwissProt Accession No. P11457. For the fusion polypeptide according to the invention, the signal sequence of E. coli Skp (aa 1-20) is removed in order to make sure that the target molecule is produced and retained in the cytosol of the overproducing prokaryotic host. Preferably, the mature form of E. coli Skp , i.e. aa 21-161 of the sequence listed below is used.
SEQ ID NO. 3 represents the complete E. coli SlyD amino acid sequence (196 amino acid residues) which is also accessible via ID P0A9K9 in the SwissProt database. For the fusion polypeptide according to the invention, preferably a C-terminally truncated version of E. coli SlyD spanning amino acid residues 1-165 of the sequence listed below is used.
SEQ ID NO. 4 shows the amino acid sequence of the glycine-rich spacer (comprising triple glycine units separated by a serine) that can be used as a flexible, soluble and protease-resistant spacer or linker between several chaperone moieties.
SEQ ID NO. 5 shows the amino acid sequence of the octa-histidine tag or “His-tag” (comprising eight histidine units) that can be added to the C-terminal end of a protein to allow Ni-NTA assisted protein purification.
SEQ ID NO: 6 shows the complete amino acid sequence of FkpA (270 aa), accessible also via the SwissProt database Accession No. P45523. For the fusion polypeptide according to the invention the signal sequence of E. coli FkpA (aa 1-25) is removed in order to make sure that the target molecule is produced and retained in the cytosol of the overproducing prokaryotic host. Preferably, the mature form of E. coli FkpA , i.e. aa 26-270 of the sequence listed below is used.
SEQ ID NO. 7 shows the complete amino acid sequence (149 amino acids) of E. coli SlpA, taken from the SwissProt database accession no. P0AEMO.
SEQ ID NO. 8 shows the amino acid sequence of Pasteurella multocida SlyD (full length) according to Swiss Prot ID: Q9CKP2
SEQ ID NO. 9 shows the amino acid sequence of Skp-tandem-SlpA or Skp-SlpA-SlpA. The glycine-rich spacer region (underlined) between the Skp and SlpA units has been added to enable maximal flexibility of the fusion partners and to make sure that the Skp units may form ordered trimers without any interference of the C-terminally fused SlpA proteins. An additional C-terminal hexa-histidine tag has been added for purification purposes. SEQ ID NO. 9 comprises amino acid residues 2-148 of E. coli SlpA (complete molecule cf. SEQ ID NO. 7, but lacking the N-terminal methionine and the C-terminal alanine) in tandem form, i.e. two E. coli SlpA (2-148) units in a row.
GGSGGGSGGG SGGGSESVQS NSAVLVHFTL
In commercially available immunoassays using rare reagents that contain SlyD fusion modules usually chemically cross-linked chaperone molecules such as tandem SlyD (two molecules of SlyD, linked via a short peptidic sequence, polymerized by means of glutardialdehyde) are added for reduction of interferences. As mentioned in the background section, due to the production process these chemically cross-linked additives are rather heterogeneous and cannot be provided in a strictly reproducible way with satisfying yields.
Although the use of polypeptide fusion proteins wherein chaperones are fused to specific target antigen sequences has been described in great detail before (see background of the invention) prior art is silent about how to overcome interferences in immunoassays. WO 2003/000878 describes FkpA as a chaperone that exerts its function in form of oligomers that can be fused to target polypeptide sequences. EP 1982993 discloses fusion polypeptides comprising at least one multimerization domain and a plurality of copies of an epitope segment from a pathogen. These polypeptides are applied as specific antigenic target sequences. However, the problem of eliminating interferences due to cross-reacting antibodies that bind to the chaperone modules and thus cause false positive results has hitherto not been addressed.
Surprisingly, by fusing a chaperone multimerization domain to at least one molecule of SlyD or SlpA we have been able to produce highly effective anti-interference agents. On top of that, in certain sera a significant background signal suggestive of a positive result cannot be minimized or suppressed effectively by the chemically cross-linked prior art reagent whereas the fusion polypeptide according to the invention is able to suppress the high background signal by reducing the interference. When added as an anti-interference agent to samples, none of the true positive samples are significantly affected, i.e. true positive samples comprising anti-HIV antibodies are invariably detected as positive samples.
Unexpectedly, by fusing a chaperone multimerization domain to at least one molecule of SlyD or SlpA we have been able to produce a well-folded, highly soluble artificial chimeric protein which forms regular trimers. It is far from self-evident that fusion of two chaperones via a flexible linker segment yields a soluble and functional protein in which the constituents largely maintain their genuine properties. Since chaperones such as Skp, SlyD and SlpA possess polypeptide binding sites which have been evolved to recognize and reversibly bind hydrophobic protein surfaces, and since these polypeptide binding sites are, themselves, hydrophobic by nature, it would have been very likely that the polypeptide binding sites of the two chaperones bind and saturate each other, leading to locked complexes with limited value for epitope-presenting purposes. It also would have been conceivable that fusion of two (sterically demanding) SlyD units to the C-terminal end of Skp compromises the structural integrity of Skp and, possibly, abolishes the intrinsic trimerization of Skp.
However, what we find is that Skp-SlyD, Skp-SlyD-SlyD and Skp-SlpA-SlpA indeed form regular trimers which are highly stable and soluble and which show—even though they are oligomeric—largely reversible folding behavior. It also would have been conceivable that the solubility of the artificial fusion polypeptide (i.e. the combination of two unrelated chaperones) turns out to be poor due to the high effective concentration of the hydrophobic polypeptide binding regions which are brought into close proximity by covalent fusion of Skp and SlyD or SlpA, respectively. Surprisingly to us, the converse is true: Skp-SlyD-SlyD possesses an outstanding solubility and it can be concentrated to >100 mg/ml (in 50 mM potassium phosphate pH 7.0, 250 mM KCl, 0.5 mM EDTA) without any tendency to aggregate. When Skp-SlyD-SlyD from such a concentrated solution is subjected to analytical gel filtration on a Superdex 200 column, the fusion polypeptide elutes in a single symmetric peak pointing to a perfectly soluble and stable trimer.
Skp-SlyD-SlyD is not only highly soluble, but its refolding behavior is also reversible. This is highlighted by the fact that we were able to elaborate a purification protocol which includes the step of matrix-coupled refolding: The target molecule is bound in an unfolded form to a Ni-NTA support via its C-terminal octa-histidine tag. While it remains bound to the solid support, it is refolded by a simple buffer change. Subsequently, the refolded protein is eluted by an imidazole pulse. Surprisingly to us, upon imidazole elution, the eluted Skp-SlyD-SlyD monomers form soluble trimers almost quantitatively. To us, it is astounding that the matrix-coupled refolding of a trimeric protein such as Skp-SlyD-SlyD works so well and with impressively high yields. We find that the same holds true for Skp-SlpA-SlpA: very similar to Skp-SlyD-SlyD, Skp-SlpA-SlpA can be obtained in large amounts from an E. coli overproducing strain and it can be renatured in a well-ordered trimeric form by matrix-assisted refolding. And, quite similar to its SlyD counterpart, Skp-SlpA-SlpA is highly soluble and possesses very favourable physicochemical properties.
According to the invention the obtained fusion polypeptide—unlike the fusion polypeptides described in prior art—is free of further target polypeptide antigenic sequences as the fusion polypeptide is used for interference reduction or protein stabilization. It is not applied as a specific antigenic polypeptide for binding to analyte molecules such as antibodies. It is important that the analyte present in the sample—like e.g. antibodies against a pathogen—does not bind to the fusion polypeptide of the invention. Therefore target polypeptide sequences like antigen sequences derived from mammalian pathogens like viruses, bacteria, single-cell or multi-cell parasites are not part of the fusion polypeptide.
The fusion polypeptide according to the invention can be obtained in a reproducible way in homogeneous fractions with high yields. It possesses a well-defined epitope density which is necessary and sufficient to recognize, bind and quench interference factors of the IgG and IgM type. The fusion polypeptide is able to suppress false positive reactions in an immunoassay in a way that is not only equivalent but superior to the chemically produced reagent of prior art. Its production process is straightforward, simple and easy to standardize and inevitably results in high yields of a well-defined homogeneous fusion polypeptide with outstanding anti-interference properties.
According to the invention the fusion polypeptide preferably contains one multimerization domain. A multimerization domain is a domain that mediates and supports non-covalent association of several protein subunits containing that very multimerization domain. For example, a dimerization domain is a domain that triggers association of two subunits, a trimerization domain is a domain that supports non-covalent association of three subunits and so on.
The second part of the fusion polypeptide is at least one molecule of SlyD, preferably E. coli SlyD, but SlyD molecules from other organisms, such as, e.g., Pasteurella multocida SlyD (cf. SEQ ID NO. 8) can also be used. Further preferred is a fusion polypeptide wherein one molecule of Skp is fused to two adjacent molecules of SlyD. In another embodiment of the invention other monomeric chaperones such as SlpA are also suitable as fusion partners which are fused to the single multimerization domain.
A further aspect of the invention is the use of the fusion polypeptides described above for reduction of interferences or for minimizing false positive results. The fusion polypeptide of the invention can be added to the immunoassay admixture (comprising sample and a binding partner specifically binding to the analyte in the sample) either before, at the same time or after said specific binding partner is added to the sample. Preferably, the fusion polypeptide is added to the test reagents before the body fluid sample containing the analyte, e.g. an antibody, is brought into contact with the specific binding partners (in this case the specific binding partners would be antigens).
Various formats and principles of immunoassays for detecting analytes and different modes of detection have been widely described and are familiar to a person skilled in the art. Of particular interest are immunoassays in which the analyte is an antibody. Preferably the immunoassay according to the invention detects antibodies against mammalian viral or bacterial pathogens such as e.g. hepatitis A, B or C virus, HIV (human immunodeficiency virus), HSV (herpes simplex virus), HTLV (human T-cell leukemia virus), EBV (Epstein-Barr virus), Rubella virus, CMV (cytomegalovirus), Treponema pallidum or Borrelia burgdorferi.
The invention is further illustrated in the examples section.
Cloning and Purification of Skp/SlyD Chaperone Fusion Polypeptides
Cloning of Expression Cassettes
In order to generate a suitable expression construct, an expression cassette encoding EcSkp-EcSlyD-EcSlyD was ligated into expression plasmid pQE80L (Qiagen, Hilden, Germany) by a two-step cloning strategy.
The sequence of the E. coli Skp (EcSkp) was retrieved from the SwissProt database (SwissProt ID P0AEU7). In a first step, a synthetic gene comprising the Shine-Dalgarno sequence from expression vector pQE80L, the coding sequence of the mature Skp chaperone amino acids 21-161 (the signal peptide spanning amino acid residues 1-20 were omitted, the ATG start codon (methionine) was added in frame), with a part of a glycine-rich linker region as well as suitable recognition sites for restriction endonucleases EcoRI (5′ end) and BamHI (3′ end), was purchased from Sloning (Vaterstetten, Germany). The synthetic 489 bp DNA fragment was hydrolyzed with the respective restriction endonucleases and ligated into the EcoRlIBamHI opened expression vector pQE80L under control of a T5 promoter (PT5).
Secondly, a further synthetic gene encoding two E. coli SlyD units (EcSlyD, residues 1-165, SwissProt accession no. P0A9K9) connected via a glycine-rich linker region and encompassing part of a further linker region at the N-terminus as well as an octa-His-tag linked with a GGGS motif to the C-terminus was likewise purchased from Sloning (Vaterstetten, Germany). BamHI and HindIII restriction sites were added at the 5′ and 3′ ends of this cassette, respectively. Genes and restriction sites were designed to enable the in frame fusion of the EcSlyD-EcSlyD part to the 5′ end of the EcSkp part by simple ligation. Therefore, the 1146 bp spanning fragment was hydrolyzed with restriction endonucleases BamHI and HindIII and ligated into the BamHI/HindIII opened, Skp containing vector pQE80L.
After ligation, competent cells of E. coli XL1Blue (Stratagene, La Jolla, Calif., USA) were transformed with the respective DNA. After plasmid preparation from suitable transformants, the correctness of the expression construct was reconfirmed by sequence analysis. The resulting expression plasmid has been named pQE80Skp-diSlyD.
The drawing below displays a scheme of the full length fusion polypeptide EcSkp-EcSlyD-EcSlyD comprising one E. coli Skp chaperone unit and two E. coli SlyD chaperone units connected by glycine rich linker regions and followed by a C-terminal octa-His-tag to allow Ni-NTA assisted protein purification.
The complete amino acid sequence of the desired fusion polypeptide is shown in SEQ ID No. 1.
The expression cassettes encoding the iterative SlyD constructs (SlyD, SlyD-SlyD, SlyD-SlyD-SlyD . . . ) have been cloned as described in Biochemistry (2006) 45, 20-33. The expression cassette encoding the Skp-SlpA-SlpA fusion polypeptide has been generated according to Scholz et al., J. Mol. Biol. (2005) 345, 1229-1242.
Recombinant Expression of EcSkp-EcSlyD-EcSlyD in an E. coli Host
In order to obtain the putative anti-interference polypeptide in sufficient amounts, EcSkp-EcSlyD-EcSlyD was recombinantly expressed in E. coli. For this purpose, competent cells of E. coli BL21 Codon+ (Merck (Novagen®), Darmstadt, Germany) were transformed with the generated expression construct pQE80Skp-diSlyD.
50 mL of SB medium (32.0 g tryptone, 20.0 g yeast extract, 5.0 g NaCl, ad 1000 mL A. dest.) supplemented with ampicillin (100 μg/mL) were inoculated with a single colony harboring the pQE80Skp-diSlyD plasmid and incubated over night at 37° C. (250 rpm). Subsequently, 1.5 L of SB medium (+100 μg/mL ampicillin) was inoculated with the overnight culture up to an O.D.600 of ˜0.5. At an O.D.600 of ˜3.0, cytosolic overexpression was induced by adding 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) to the culture. Four hours after induction, cells were harvested by centrifugation (20 min at 6000 g) and stored at −20° C.
Aliquots of 0.4 O.D.600 were taken before and four hours after induction and whole cell extracts were tested for expression of EcSkp-EcSlyD-EcSlyD by SDS-PAGE analysis. Overproduction of the target molecule was found to be highly abundant.
Overexpression of the SlyD and SlpA fusion proteins was carried out as described in Scholz et al., J. Mol. Biol. (2005) 345, 1229-1242 and Scholz et al., Biochemistry (2006) 45, 20-33.
Purification of Skp/SlyD/SlpA Polypeptide Fusions
The Skp/SlyD polypeptide fusions and the SlyD and SlpA fusion proteins were purified by using virtually identical protocols. For cell lysis, the frozen pellet was resuspended in chilled 50 mM sodium phosphate pH 8.0, 7.0 M GdmCl, 5 mM imidazole and the suspension was stirred for at least 2 h on ice to complete cell lysis. After centrifugation and filtration (0.45 μm/0.2 μm), the crude lysate was applied onto a Ni-NTA column equilibrated with the lysis buffer including 5.0 mM TCEP. The subsequent washing step was tailored for the respective target protein and ranged from 5 to15 mM imidazole (in 50 mM sodium phosphate pH 8.0, 7.0 M GdmC1, 5.0 mM TCEP). At least 10-15 volumes of the washing buffer were applied. Then, the GdmCl solution was replaced by 50 mM potassium phosphate pH 8.0, 100 mM KCl, 10 mM imidazole, 5.0 mM TCEP to induce conformational refolding of the matrix-bound protein. In order to avoid reactivation of copurifying proteases, a protease inhibitor cocktail (Complete® EDTA-free, Roche) was included in the refolding buffer. A total of 15-20 column volumes of refolding buffer were applied in an overnight reaction. Then, both TCEP and the Complete® EDTA-free inhibitor cocktail were removed by washing with 3-5 column volumes 50 mM potassium phosphate pH 8.0, 100 mM KCl, 10 mM imidazole. Subsequently, the imidazole concentration—still in 50 mM potassium phosphate pH 8.0, 100 mM KCl—was raised to 60-70 mM for the EcSkp-EcSlyD fusion proteins, to 50 mM for EcSkp-EcSlpA-EcSlpA and to 30 mM for the SlyD fusion polypeptides in order to remove unspecifically bound protein contaminants. The native protein was then eluted by 500 mM imidazole in the same buffer. Protein-containing fractions were assessed for purity by Tricine-SDS-PAGE and pooled. Finally, the proteins were subjected to size-exclusion-chromatography (Superdex HiLoad, Amersham Pharmacia) and the protein-containing fractions were pooled and concentrated to 10-20 mg/ml in an Amicon cell (YM10). After the coupled purification and refolding protocol, protein yields of roughly 15-25 mg could be obtained from 1 g of E. coli wet cells, depending on the respective target protein.
Spectroscopic Measurements
Protein concentration measurements were performed with an Uvikon XL double-beam spectrophotometer. The molar extinction coefficients (ε280) were determined by using the procedure described by Pace (1995), Protein Sci. 4, 2411-2423. For EcSkp-EcSlyD-EcSlyD, a molar extinction coefficient (εM280) of 13410 M−1 cm−1 was used, for EcSkp-EcSlyD a molar extinction coefficient (εM280) of 7450 M−1 cm−1 was used. For EcSkp-EcSlpA-EcSlpA, a molar extinction coefficient (εM280) of 4470 M−1 cm−1 was used. For the repetitive E. coli SlyD constructs, SlyD, SlyD-SlyD, SlyD-SlyD-SlyD, SlyD-SlyD-SlyD-SlyD and SlyD-SlyD-SlyD-SlyD-SlyD, molar extinction coefficients of 5960 M−1 cm−1, 11920 M−1cm−1, 17880 M−1 cm−1, 23840 M−1 cm−1 and 29800 M−1 cm−1 were used.
The anti-interference activity of the SlyD polypeptide fusion proteins was assessed in an automated Elecsys® 2010 analyzer (Roche Diagnostics GmbH). Elecsys® is a registered trademark of the Roche group. Measurements were carried out in the double antigen sandwich format.
Signal detection in Elecsys® 2010 is based on electrochemoluminescence. The biotin-conjugate (i.e. the capture-antigen) is immobilized on the surface of a streptavidin coated magnetic bead whereas the detection-antigen bears a complexed Ruthenium cation (switching between the redox states 2+ and 3+) as the signaling moiety. In the presence of a specific immunoglobulin analyte, the chromogenic ruthenium complex is bridged to the solid phase and emits light at 620 nm after excitation at a platinum electrode. The signal output is in arbitrary light units.
The recombinant anti-interference SlyD polypeptides were assessed in a double antigen sandwich (DAGS) immunoassay format. To this end, FkpA-FkpA-gp41 and SlyD-SlyD-gp41 as disclosed in EP 1 402 015 were used as biotin and ruthenium conjugates, respectively, to specifically detect anti-gp41 antibodies in human sera. gp41 is the immunodominant antigen of HIV, and soluble variants of the gp41 ectodomain—as disclosed in EP 1 402 015—are invaluable tools for the detection of HIV infections. FkpA-FkpA-gp4l-biotin and SlyD-SlyD-gp41-ruthenium were used in R1 (reagent buffer 1) and R2 (reagent buffer 2) at concentrations of 750 ng/ml each.
In a first experiment, Trina sera negative for HIV were assessed with the aforementioned DAGS immunoassay setup. In order to get a hint to the incidence rate of false positives, the assessment was performed in the absence and in the presence of SS-Helix(GDA,P), a GDA-crosslinked soluble heterogeneous SlyD polymer which is used as an anti-interference substance. SS-Helix (GDA,P) was added to R1 (reagent buffer 1 containing the biotin conjugate) in large excess amounts (25 μg/ml). 60 μl R1 (reagent buffer 1, biotin conjugate and anti-interference SlyD polymer), 60 μl R2 (reagent buffer 2, ruthenium conjugate), 30 μl sample (human serum) and 50 μl bead suspension are then mixed and incubated to yield a reaction volume of roughly 200 μl.
Tables 1a-c (
Tables 2a-c (
In Tab. 2b (
To critically challenge this assumption, we performed further anti-interference studies shown in table 2c (
Table 2c (
In brief, Tab. 2 (
Tab. 3 (
The anti-interference activity of EcSkp-EcSlpA-EcSlpA was assessed in an automated Elecsys® 2010 analyzer (Roche Diagnostics GmbH). Elecsys® is a registered trademark of the Roche group. Measurements were carried out in the double antigen sandwich format.
Signal detection in Elecsys® 2010 is based on electrochemoluminescence (for detailed explanation see example 3).
The recombinant EcSkp-EcSlpA-EcSlpA polypeptide was assessed in a double antigen sandwich (DAGS) immunoassay format. To this end, PmSlyD-mgG2 and EcSlpA-mgG2 as disclosed in EP 2 127 678 were used as biotin and ruthenium conjugates, respectively, to specifically detect anti-HSV-2 antibodies in human sera. Mature glycoprotein G2 (mgG2) is an immunodominant antigen of herpes simplex virus 2 (HSV-2), and soluble variants thereof—as disclosed in EP 2 127 678—are invaluable tools for the detection of HSV-2 infections. PmSlyD-mgG2-biotin and EcSlpA-mgG2-ruthenium conjugates were used in R1 (reagent buffer 1) and R2 (reagent buffer 2) at concentrations of 300 ng/ml each.
Anti-HSV-2 negative sera, anti-HSV-2 positive sera and anti-HSV-2 false positive sera (i.e. interference sera) were assessed with the aforementioned DAGS immunoassay setup. The assessment was performed in the absence and in the presence of EcSlpA-EcSlpA (GDA,P), a soluble heterogeneous GDA-crosslinked EcSlpA polymer which is used as an anti-interference substance in the anti-HSV-2 immunoassay. EcSlpA-EcSlpA (GDA,P) serves the role of an anti-interference benchmark: it constitutes the conventional anti-interference additive that has been generated by chemical crosslinking of an EcSlpA-EcSlpA polypeptide and that is well-suited to improve the specificity of immunoassays based on EcSlpA fusion antigens. The anti-interference additives under scrutiny were added to both R1 (reagent buffer 1, containing the biotin conjugate) and to R2 (reagent buffer 2, containing the ruthenium conjugate) in large excess amounts (10 μg/ml each). 70 μl R1 (reagent buffer 1, biotin conjugate and anti-interference EcSlpA polymer), 70 μl R2 (reagent buffer 2, ruthenium conjugate and anti-interference EcSlpA polymer), 20 μl sample (human serum) and 40 μl bead suspension are then mixed and incubated to yield a reaction volume of roughly 200 μl.
Even though two different chaperones, such as PmSlyD (i.e., SlyD from Pasteurella multocida) and EcSlpA (i.e., SlpA from E. coli), are used on the two sides of the double antigen sandwich immunoassay, significantly elevated signals are rather frequent in a panel of human sera, for which HSV-2 infections have clearly been ruled out. The reason for this finding is that the fusion partners PmSlyD and EcSlpA, although from different organisms, are related molecules belonging to the FKBP family of chaperones and sharing a highly conserved FKBP domain. It is probably via this shared motif that the immunological cross-reaction takes place, evoking high signals and thus pretending a positive outcome in the anti-HSV-2 assay. Addition of the chemically polymerized anti-interference substance, EcSlpA-EcSlpA(GDA,P), to the assay mixture reduces the elevated signals to normal negatives. Table 4 (
When EcSkp-EcSlpA-EcSlpA is added to the assay, we find that the false positive signals are reduced to the signal level of HSV-2 negative sera as well. Indeed, EcSkp-EcSlpA-EcSlpA turns out to be at least as efficient as the chemically polymerized EcSlpA-EcSlpA (GDA,P) in its anti-interference capacity. Obviously, the epitope density of Skp-EcSlpA-EcSlpA is high enough to efficiently bind to and quench interference factors which presumably belong to the IgM type of immunoglobulins. These interference sera are characterized in that they do not respond to the addition of monomeric anti-interference additives (see tab. 4/
Near-UV CD spectra were recorded with a Jasco-720 spectropolarimeter with a thermostatted cell holder and converted to mean residue ellipticity. The buffer was 50 mM potassium phosphate pH 7.5, 250 mM KC1, 0.5 mM EDTA. The pathlength was 0.2 cm the protein concentration was 8.2 mg/ml (147 μM monomer, corresponding to 49 μM trimer). The range was 250-330 nm, the band width was 1.0 nm, the scanning speed was 20 nm/min at a resolution of 0.5 nm and the response was 1 s. In order to improve the signal-to-noise ratio, spectra were measured nine times and averaged.
Circular dichroism spectroscopy (CD) is the method of choice to assess both the secondary and the tertiary structure of proteins. Ellipticity in the aromatic region (250-330 nm) reports on tertiary contacts within a protein (i.e., the globular structure of a regularly folded protein) and is considered as the fingerprint region of a native-like fold (conformation).
Near UV CD spectra of Skp-SlyD-SlyD were monitored to address the question whether the fusion protein adopts an ordered conformation after the matrix-coupled refolding procedure which is the crucial step in the purification process. The answer is quite clear-cut: the near UV CD signal of Skp-SlyD-SlyD unequivocally reports an orderly tertiary structure of the fusion polypeptide. The aromatic residues of Skp-SlyD-SlyD are obviously embedded in the lipophilic protein core and thus experience asymmetric surroundings which strongly points to a native-like conformation of both Skp and SlyD within the fusion construct (
In order to address the question whether the thermally induced unfolding of Skp-SlyD-SlyD is reversible, melting curves were monitored in the near UV region at a detection wavelength of 280 nm. The temperature range was 20-65° C., the band width was 1.0 nm, the temperature slope was 1° C./min and the response was 4 s (see
The thermally-induced unfolding was monitored at 280 nm (which is the wavelength of the maximal signal amplitude for Skp-SlyD-SlyD). Upon heating, the non-covalent contacts which stabilize the native conformation of the Skp-SlyD-SlyD molecule become loose and finally break down. This thermally induced unfolding is reflected in a decrease in the CD signal as shown in
We found very similar results for Skp-SlpA-SlpA: just like Skp-SlyD-SlyD, Skp-SlpA-SlpA exhibits a marked CD signal in the near UV region (250-330 nm, signal maximum at 277 nm), pointing to a well-ordered conformation after the matrix-coupled refolding process. By means of thermal transitions (monitored at 277 nm) we observed that Skp-SlpA-SlpA retains its native-like conformation at temperatures up to 55° C. Furthermore, the CD signal of the native molecule is largely restored after a thermal unfolding/refolding cycle (20° C./65° C./20° C.) as illustrated by
In conclusion, Skp-SlyD-SlyD and Skp-SlpA-SlpA possess robust folding properties which are outstanding for molecules with this degree of complexity and which are highly desirable for modules that serve as anti-interference or generally stabilizing components of an immunoassay.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12006298.9 | Sep 2012 | EP | regional |
This application is a continuation of International Patent Application No. PCT/EP2013/068269 filed Sep. 4, 2013, and claims priority to EP Patent Application No. 12006298.9 filed Sep. 6, 2012, the disclosures of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2013/068269 | Sep 2013 | US |
| Child | 14640263 | US |
