Magnetic particle-binding peptides

Abstract

The present invention relates to a peptide consisting of a sequence of 5 to 30, preferably 6 to 12, most preferably 10 to 12 amino acids, wherein (a) at least ? of said amino acids have a functional group or side chain which is negatively charged at neutral pH; (b) amino acids which do not have a functional group or side chain which is negatively charged at neutral pH, if present, meet one or both of requirements (i) and (ii): (i) none of them has a functional group or side chain which is positively charged at neutral pH; and (ii) at least one of them has a side chain which does not bear a net charge at neutral pH or which has a functional group or side chain that does not bear a net charge at neutral pH.

Claims

1. A peptide consisting of a sequence of 5 to 30 amino acids, wherein (a) at least ? of said amino acids have a functional group or side chain which is negatively charged at neutral pH; (b) amino acids which do not have a functional group or side chain which is negatively charged at neutral pH, if present, meet one or both of requirements (i) and (ii): (i) none of them has a functional group or side chain which is positively charged at neutral pH; and (ii) at least one of them has a side chain which does not bear a net charge at neutral pH or which has a functional group or side chain that does not bear a net charge at neutral pH.

2. The peptide of claim 1, wherein the fraction of amino acids which have a functional group or side chain which is positively charged at neutral pH is (a) in the range between zero and 0.2; or (b) greater than 0.2.

3. The peptide of claim 1 or 2, wherein said at least one amino acid as defined in claim 1(b)(ii) is present and is flanked on either side by at least one amino acid as defined in claim 1(a).

4. The peptide of claim 1, wherein said peptide is capable of binding to a surface comprising iron oxide.

5. The peptide of claim 1, wherein (a) at least one amino acid has a side chain which is negatively charged at neutral pH are selected from Asp and Glu; and/or (b) at least one amino acid, to the extent present, which has a side chain which does not bear a net charge at neutral pH are selected from Gly, Ser, Ala, Asn, Cys, Gln, His, Ile, Leu, Met, Phe, Pro, Thr, Trp, Tyr, Val and selenocystein.

6. A molecule which is a polypeptide or protein, said polypeptide or protein comprising at least one sequence of a peptide as defined in any one of the preceding claims, wherein the sequence(s) in said polypeptide or protein which do(es) not comprise said at least one sequence of a peptide are heterologous with respect to said sequence of a peptide.

7. A molecule covalently bound to at least one sequence of a peptide as defined in claim 1, wherein the covalent bond is not a main chain peptide bond, and wherein said molecule is a polypeptide or protein or a nucleic acid.

8. A surface comprising iron oxide, preferably a magnetic nanoparticle, which is bound to at least one peptide or molecule as defined in claim 1.

9. A method of separating a molecule as defined in claim 6 from a sample, said method comprising: (a) contacting said sample with a surface comprising iron oxide, preferably magnetic nanoparticles, under conditions allowing binding of said molecule to said surface; and (b) separating said surface from said sample; thereby separating said molecule from said sample.

10. A method of separating an analyte from a sample, said analyte being capable of binding to a molecule as defined in claim 6, said method comprising: (a) bringing into contact said sample, said molecule and a surface comprising iron oxide, preferably magnetic nanoparticles, under conditions allowing (i) binding of said molecule to said surface and (ii) binding of said analyte to said molecule; and (b) separating said surface from said sample; thereby separating said analyte from said sample.

11. The method of claim 9 or 10, further comprising (c) washing said surface; and/or (d) (i) dissociating said molecule from said surface; (ii) optionally followed by removing said surface from the result of (i).

12. The method of claim 11, wherein said dissociating in step (d)(i) is performed by adding (a) a carboxylic acid, preferably a carboxylic acid comprising two or three carboxylic groups; and/or (b) an inorganic oxo acid.

13. A method of determining presence or absence of an analyte in a sample suspected of comprising said analyte, said method comprising (a) bringing into contact said sample, said molecule and a surface comprising iron oxide, under conditions allowing (i) binding of said molecule to said surface and (ii) binding of said analyte to said molecule, and (b) separating said surface from said sample, thereby separating said analyte from said sample; and optionally at least one of steps (c), (d)(i), (d)(ii) and (e) as defined in claim 11, thereby obtaining a mixture, and (f) analyzing said mixture for the presence of said analyte.

14. A kit comprising (a) (i) a peptide as defined claim 1; (ii) a molecule which is a polypeptide or protein, wherein the sequence(s) in said polypeptide or protein do(es) not comprise said at least one sequence of a peptide are heterologous with respect to said sequence of the peptide as defined in claim 1; and/or (iii) a nucleic acid encoding a peptide of (i) or a molecule of (ii), wherein said molecule is a polypeptide or protein.

15. The kit of claim 14, further comprising at least one of (b), (c), (d) and (e): (b) a surface comprising iron oxide; (c) a first solution or constituents for preparing said first solution, said first solution being capable of establishing conditions which allow binding of said peptide and/or said molecule to said surface; (d) a second solution or constituents for preparing said second solution, said second solution being capable of establishing conditions which allow dissociating said peptide and/or said molecule from said surface; and (e) instructions for use of said kit, instructing separating a molecule which is a polypeptide or protein, said polypeptide or protein comprising at least one sequence of a peptide as defined in any one of the preceding claims, wherein the sequence(s) in said polypeptide or protein which do(es) not comprise said at least one sequence of a peptide are heterologous with respect to said sequence of a peptide from a sample, said method comprising: (i) contacting said sample with a surface comprising iron oxide, preferably magnetic nanoparticles, under conditions allowing binding of said molecule to said surface; and (ii) separating said surface from said sample.

Description

[0083] The Figures show:

[0084] FIG. 1: Binding scores of magnetic nanoparticles on peptides at pH 7.4 and pH 8 in Tris buffered saline with 0.25% Tween 20 (T-TBS). The particle suspensions were disaggregated in an ultrasound bath for 15 min before incubation with the membrane. The results are the averages of two membranes tested in the same experiment. The horizontal line indicates the noise level in this experiment.

[0085] FIG. 2: Binding affinities of magnetic nanoparticles to peptides in Citrate buffered saline pH 6 with 0.25% Tween 20 (T-CBS). The particle suspensions were disaggregated in an ultrasound bath for 15 min before incubation with the membrane. The horizontal line stands for the background noise level.

[0086] FIG. 3: Binding scores of MNPs on peptide spots after incubation in T-TBS pH 7.4 for 1 h and after transfer of membrane to T-CBS pH 6. (A) The noise level is represented by the horizontal line. (B) Image of a membrane with spots that bound to MNPs being marked.

[0087] FIG. 4: In accordance with art-established methods, prior to any application, magnetic nanoparticles have to be functionalized in a tailored manner. In the figure, functionalization of the nanoparticle requires coating thereof with the molecules displayed in dark grey.

[0088] FIG. 5: Purification of polypeptides or proteins tagged with peptides of the present invention with magnetic nanoparticles. Peptides of the present invention are fused to the polypeptide or protein of interest and MNPs are added to the initial mixture. MNPs bind to the polypeptide or protein and can be separated by using a magnet. Upon changing the conditions, the polypeptide or protein can be separated from the magnetic nanoparticles. The magnetic nanoparticles in turn can be separated and re-used again.

[0089] FIG. 6: An antibody having peptides in accordance with the present invention fused to its F.sub.c portion binds to magnetic nanoparticles. Antibody-loaded nanoparticles can be used to separate molecules comprising the cognate epitope from a mixture. When peptides in accordance with the present invention are used which bind reversible to magnetic nanoparticles, the antibody-antigen complex can be detached from the magnetic nanoparticle by changing the conditions. As in the preceding figures, magnetic nanoparticles can be re-used.

[0090] FIG. 7: Interaction data for peptides on a PEPperCHIP?-Array with magnetic nanoparticles in ddH2O at pH 4. Data are averages for duplicates of the same experiment.

[0091] FIG. 8: Scores for the binding of peptides to magnetic nanoparticles in Tris buffered saline (T-TBS) at pH 7.4 on cellulose membranes from Intavis. The data represent averages of two experiments.

[0092] FIG. 9: Adsorption isotherms of octa-glutamic acid on magnetite nanoparticles (1 g L.sup.?1) at different pH (top) and in different buffers at pH 7 (bottom), all without Tween. Adsorption isotherms at pH 4.5 and 7 are fitted with a Langmuir function, while the isotherms at pH 9 and 10 are fitted with a Freundlich function.

[0093] FIG. 10: Adsorption isotherms of tagged green fluorescent protein (GFP) on magnetic nanoparticles in Tris buffered saline (TBS) at pH 7 with 0.1% Tween (T-TBS).

[0094] FIG. 11: Magnetic separation of Glu.sub.6-tagged protein from bacterial lysate in TBS at pH 7 using a high gradient magnetic separator. Elution has been effected with citrate buffered saline (CBS) at pH 7.

[0095] The Examples illustrate the invention.

EXAMPLE 1

Materials and Methods

Synthesis of Magnetic Nanoparticles

[0096] The iron oxide nanoparticles used for this study were synthesized by co-precipitation of Fe.sup.2+ and Fe.sup.3+ in alkaline aqueous solutions adapted to the procedure described by Roth et al. (Roth, H.-C., et al. (2015): Influencing factors in the CO-precipitation process of superparamagnetic iron oxide nano particles: A model based study, Journal of Magnetism and Magnetic Materials 377, pp. 81-89). Briefly, 21.2 g of FeCl.sub.3?6 H.sub.2O and 8.29 g of FeCl.sub.2?4 H.sub.2O were dissolved in 200 mL of deionized, degassed water which equals a Fe(III):Fe(II) ratio of 1.88:1. This iron chloride solution was added to 1 L of a 1 M solution of NaOH prepared with deionized, degassed water stirring at 250 rpm in a reaction vessel. The reaction mixture was kept under nitrogen atmosphere at 25? C. and stirred for 30 more minutes before the nanoparticles that had formed were washed several times with deionized water until the conductivity of the MNP solution was below 200 ?S cm.sup.?1. In order to separate the particles from the washing water the mixture was placed on a NdFeB permanent magnet. FeCl.sub.3?6 H.sub.2O and sodium hydroxide were purchased from AppliChem GmbH, Germany in the highest purity available. FeCl.sub.2?4 H.sub.2O extra pure was obtained from Merck KGaA, Germany.

Zetapotential Measurements

[0097] For zetapotential measurements, a suspension of 0.4 g/L magnetic nanoparticles in buffer was sonicated for 15 minutes. In a Beckman Coulter Delsa Nano C, the zetapotential was determined at 25? C. three times with 10 accumulation times at 5 different positions in a flow cell each at 60 V with a pinhole of 50 ?m.

Magnetic Nanoparticle Binding Assay

[0098] In order to determine the binding between peptides and magnetic nanoparticles (MNPs) CelluSpot peptide arrays from Intavis with 5 to 10 nmol of peptides per spot were used. The cellulose membrane on which the peptides had been synthesized by the manufacturer was conditioned with 1 mL of methanol in order to rehydrate hydrophobic peptides (Golemis, E. and Adams, P. D. (2005): Protein-protein interactions. A molecular cloning manual. 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). The buffers employed were T-CBS, T-PBS or T-TBS which is 50 mM citrate, phosphate or tris, respectively, with 0.25% Tween 20, 137 mM NaCl and 2.7 mM KCl. The orbital shaker used for incubation was MulitBio3D from Biosan.

[0099] The array was washed three times for 10 min each with 50 mL of buffer. After washing, the membrane was incubated for 60 minutes in a MNP solution (Kuboyama, M., et al. (2012): Screening for silver nanoparticle-binding peptides by using a peptide array, Biochemical Engineering Journal 66 (0), pp. 73-77). The spots on which magnetite bound became dark grey. In order to remove unbound particles the membrane was washed three times for 10 minutes each with buffer (Golemis, E. and Adams, P. D. (2005): Protein-protein interactions. A molecular cloning manual. 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Then the cellulose membrane was dried overnight at 4? C. and an image of it was taken using a GelDoku station.

Peptidarray-Experimente

[0100] The PEPperCHIP?-Array with printed peptides has been purchased from PEPperPRINT Germany. 0.4 g/L MNP suspended in ddH2O pH 4 have been desagglomerated by ultrasonication for 4 minutes with a Branson sonifier 450D prior to a 1:10 dilution with ddH2O pH 4. This suspension was incubated on the chip with an orbital shaker for 1 h and then dipped into 1 mM Tris buffer pH 7.4. The chip was scanned with a Typhoon FLA 9500 scanner in digitalization mode with a resolution of 10 ?m and analyzed with the software PepSlideAnalyzer.

Adsorption Isotherms with Peptides

[0101] The octa-glutamic acid peptide (101 mg) was obtained from metabion international AG (Germany) as a lyophilized powder with a purity of >95% and stored at +4? C. prior to use. For the binding experiments different amounts of peptides were incubated with magnetite nanoparticles (1 g L.sup.?1) at different pH and buffer conditions for at least one hour at 25? C. under vigorous shaking. The supernatant was separated from the particles with hand magnets, decanted and analysed with an Infinite M200 Microplate Reader (Tecan Deutschland, Germany) at 230 nm.

Adsorption Isotherms with Proteins

[0102] 0.5 mL protein solution was mixed with a magnetite particle solution 1 g L.sup.?1 in a 1.5 mL Eppendorf vessel and incubated for 1 h at 25? C. and 1000 rpm in a thermomixer comfort from Eppendorf.

[0103] The particles and the supernatant were separated with a NdFeB magnet. The supernatant was centrifuged at 17000?g at 4? C. for 5 minutes in order to remove residual particles prior to analysis.

High-Gradient Magnetic Separation

[0104] The process starts with the incubation of 0.5 L cell lysate in TBS pH 7 containing Glu.sub.6-GFP with 0.5 L magnetite (2 g/L) in a feed tank. This mixture is stirred at ?900 rpm for 1 h at room temperature. Every 15 min aliquots of 2 mL are taken in order to monitor the adsorption.

[0105] After the incubation, the suspension is pumped through a magnetic separation chamber as described (Roth et al. A High-Gradient Magnetic Separator for Highly Viscous Process Liquors in Industrial Biotechnology, Chemical Engineering and Technology 39 (3) 469-476). The supernatant containing impurities and unbound protein is removed by two washing steps, each 0.5 L containing TBS pH 7. The flow direction through the chamber is changed every 200 s during the washing procedure. During the washing procedure 10 samples are taken of the outlet fractions. For the elution citrate buffered saline (CBS) at pH 7 is used which is provided in the feed tank. The particles are resuspended as the magnetic field is removed and the whole mixture is circulated through the whole apparatus with changing flow directions every 15 minutes. Aliquots are taken each 10 minutes and after 1 h the suspension is pumped through the magnetic field again in order to separate the protein from the particles. In a second step the resuspension is improved by the addition of a two phase flow where air is mixed with the buffer for 10 minutes as described (Roth et al. A High-Gradient Magnetic Separator for Highly Viscous Process Liquors in Industrial Biotechnology, Chemical Engineering and Technology 39 (3) 469-476). Aside from the two phase flow through the system, the process is similar to the first elution step.

EXAMPLE 2

Peptide Arrays

[0106] The binding behavior of magnetite nanoparticles (MNPs) to peptides was investigated using a peptide array. FIG. 1 shows that at a pH of 7.4 in Tris buffered saline, the negatively charged peptides hexa-glutamic acid (6E) and hexa-aspartic acid (6D) bound strongest to the MNPs. The second highest scores at pH 7.4 were achieved by the positive peptides hexa-arginine (6R), hexa-lysine (6K), 5RH and 5RE as well as hexa-histidine (6H) which is nearly neutral at a pH of 7.4 according to the theoretical pl of 7.21 determined by the ProtParam Tool by the Bioinformatics Resource Portal ExPASy (Bjellqvist, B., et al. (1993): The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences, Electrophoresis 14 (1), pp. 1023-1031). It is in accordance with literature that histidine residues can form complexes with metals and that carboxy groups bind them through electrostatic interaction (Kozlowski, H., et al. (2013): Specific metal ion binding sites in unstructured regions of proteins, Coordination Chemistry Reviews 257 (19-20), pp. 2625-2638). The MNP surface was shown to be slightly positively charged in zetapotential measurements at a pH of 7.4 as shown in Table 1. The point of zero charge was determined to be around 7.8 by acidometry. Hydrophobic peptides do not show any binding while the binding of polar peptides is low to moderate.

TABLE-US-00002 TABLE 1 Zetapotential of magnetic nanoparticles in different buffers and at different pHs Buffer pH Zetapotential, mV T-TBS 7.4 +3.7 8 T-PBS 6 ?27.8 7.4 8 T-CBS 6 ?34.74

EXAMPLE 3

[0107] Comparison of Homooligomers with Heterooligomers

[0108] As demonstrated in FIG. 7, heteromeric peptides with interspersed serine, glycine or glutamine residues exhibit better binding than homooligomers of glutamine or asparagines. The heteromers have a length between 9 and 12 amino acids and the fraction of residues which are negatively charged at neutral pH is at least ?.

[0109] As shown in FIG. 8, such results could be confirmed on a different array format. In particular, to the extent the fraction of cysteine, glycine or asparagines is above ?, binding drops below the binding of the corresponding homooligomers. On the other hand, if the fraction of cysteine, glycine or asparagines does not exceed ?, binding is increased as compared to the corresponding homooligomers.

[0110] FIG. 9 shows affinity constants of peptides in accordance with the present invention to magnetic nanoparticles in different buffers and at different pH values.

EXAMPLE 4

Separation Method

[0111] The data depicted in FIG. 10 demonstrate that peptides in accordance with the present invention when used in proteins to be purified allow binding to magnetic nanoparticles. In a tris buffered system, Glu.sub.6-, Glu.sub.6-, and Glu.sub.4Gly.sub.4Glu.sub.4-tagged protein binds with high affinity and high maximal loads as compared to untagged protein; see also Table 2 below.

TABLE-US-00003 TABLE 2 Binding constants of tagged proteins to MNPs in T-TBS at pH 7. Protein q.sub.max, g .Math. g.sup.?1 K.sub.D, g .Math. L.sup.?1 GFP-Glu.sub.8 0.179 ? 0.006 4E?04 ? 2E?04 GFP-Glu.sub.6 0.120 ? 0.008 4E?15 ? 3E?03 GFP-E.sub.4G.sub.4E.sub.4 0.102 ? 0.011 7E?10 ? 6E?03 GFP-Gly.sub.6 0.047 ? 0.005 1E?03 ? 1E?03 GFP no tag 8E?05 ? 1E?03 2E?16 ? 6E?01

[0112] Using a magnetic separator (as described in Roth et al., Chem. Eng. Technol. 2016, 39, 469-476) has been used to separate GFP tagged with a peptide in accordance with the invention (Glu.sub.x) from a bacterial broth using magnetic particles in TBS. CBS has been used as elution buffer. FIG. 11 displays the concentration of the protein of interest (GFP-Glu.sub.6) and the total protein concentration as a function of time.