ACTIVITY ASSAY FOR BOND FORMING ENZYMES

Abstract

Herein is reported a method for the detection of a sortase in a sample, comprising the following steps: a) incubating the sample with a first substrate comprising an immobilization tag and a second substrate comprising a detectable label, whereby in the presence of a sortase in the sample a conjugate comprising the immobilization tag and the detectable label is formed, b) immobilizing the conjugate of step a) via/using the immobilization tag to a solid phase, c) detecting the immobilized conjugate via/using the detectable label
and thereby detecting the sortase in the sample.

Claims

1.-16. (canceled)

17. A method for the detection of a bond forming enzyme in a sample, comprising the following steps: a) incubating the sample with a first substrate comprising an immobilization tag and a second substrate comprising a detectable label, whereby in the presence of a bond forming enzyme in the sample a conjugate comprising the immobilization tag and the detectable label is formed, b) centrifuging the sample, c) immobilizing the conjugate of step a) using the immobilization tag to a solid phase, d) detecting the immobilized conjugate using the detectable label, and thereby detecting the bond forming enzyme in the sample.

18. The method according to claim 17, wherein after the incubation of step a) additionally the sample is diluted between 10 times and 30 times.

19. The method according to claim 17, wherein the first substrate comprising an immobilization tag comprises a first bond forming enzyme recognition motif and the second substrate comprising a detectable label comprises a second bond forming enzyme recognition motif.

20. The method according to claim 18, wherein the first substrate comprising an immobilization tag comprises a first bond forming enzyme recognition motif and the second substrate comprising a detectable label comprises a second bond forming enzyme recognition motif.

21. The method according to claim 19, wherein the first bond forming enzyme recognition motif is a first sortase recognition motif and the second bond forming enzyme recognition motif is a second sortase recognition motif.

22. The method according to claim 20, wherein the first bond forming enzyme recognition motif is a first sortase recognition motif and the second bond forming enzyme recognition motif is a second sortase recognition motif.

23. The method according to claim 21, wherein a) the first sortase recognition motif comprises an LPXTG motif (SEQ ID NO: 01), wherein X represents any amino acid and the second sortase recognition motif comprises an oligo-glycine or oligo-alanine motif or b) the second sortase recognition motif comprises an LPXTG motif (SEQ ID NO: 01), wherein X represents any amino acid and the first sortase recognition motif comprises an oligo-glycine or oligo-alanine motif.

24. The method according to claim 22, wherein a) the first sortase recognition motif comprises an LPXTG motif (SEQ ID NO: 01), wherein X represents any amino acid and the second sortase recognition motif comprises an oligo-glycine or oligo-alanine motif or b) the second sortase recognition motif comprises an LPXTG motif (SEQ ID NO: 01), wherein X represents any amino acid and the first sortase recognition motif comprises an oligo-glycine or oligo-alanine motif.

25. The method according to claim 17, wherein the detectable label is an enzyme.

26. The method according to claim 17, wherein the immobilization tag is a first member of a specific binding pair and the solid phase is conjugated to the second member of the specific binding pair.

27. The method according to claim 19, wherein the immobilization tag is a first member of a specific binding pair and the solid phase is conjugated to the second member of the specific binding pair.

28. The method according to claim 20, wherein the immobilization tag is a first member of a specific binding pair and the solid phase is conjugated to the second member of the specific binding pair.

29. The method according to claim 17, wherein the immobilization tag is biotin and the solid phase is conjugated to streptavidin or avidin.

30. The method according to claim 26, wherein the immobilization tag is biotin and the solid phase is conjugated to streptavidin or avidin.

31. The method according to claim 27, wherein the immobilization tag is biotin and the solid phase is conjugated to streptavidin or avidin.

32. The method according to claim 28, wherein the immobilization tag is biotin and the solid phase is conjugated to streptavidin or avidin.

33. The method according to claim 29, wherein the solid phase is a multiwell plate or a bead.

34. The method according to claim 30, wherein the solid phase is a multiwell plate or a bead.

35. The method according to claim 31, wherein the solid phase is a multiwell plate or a bead.

36. The method according to claim 32, wherein the solid phase is a multiwell plate or a bead.

37. The method according to claim 17, wherein after the incubation of step a) the sample is diluted with a solution comprising iodacetamide (IAA).

38. The method according to claim 17, wherein the sample is centrifuged between 5 min and 15 min and at between 2500×g to 7500×g.

39. The method according to claim 18, wherein the sample is centrifuged between 5 min and 15 min and at between 2500×g to 7500×g.

40. The method according to claim 37, wherein the sample is centrifuged between 5 min and 15 min and at between 2500×g to 7500×g.

41. The method according to claim 17, wherein after the immobilization of step c) additionally the sample is washed at least 3 times.

42. The method according to claim 37, wherein after the immobilization of step c) additionally the sample is washed at least 3 times.

43. The method according to claim 38, wherein after the immobilization of step c) additionally the sample is washed at least 3 times.

44. The method according to claim 39, wherein after the immobilization of step c) additionally the sample is washed at least 3 times.

45. The method according to claim 40, wherein after the immobilization of step c) additionally the sample is washed at least 3 times.

46. A method for selecting a bond forming enzyme from a multitude of bond forming enzymes comprising the steps of: a) providing at least two bond forming enzymes, b) performing the method of claim 17 with the at least two bond forming enzymes, and c) selecting a bond forming enzyme detected in step b).

47. The method according to claim 46, wherein the bond forming enzymes with the highest activity is selected.

48. The method according to claim 46, wherein the bond forming enzyme is a sortase.

Description

DESCRIPTION OF THE FIGURES

[0171] FIG. 1 Effect of Sortase concentration on ligation yield. Yield was determined based on protocol for measuring sortase activity (see example 2)

[0172] FIG. 2 Effect of Sortase concentration on product formation. Yield was determined based on protocol for measuring sortase activity (see example 2)

[0173] FIG. 3 Comparison of different method setups with respect to the coefficients of variation. 1) Method as described herein; 2) Method as described herein lacking the centrifugation step; 3) Method as described herein lacking the centrifugation step and lacking the step of stopping the reaction by addition on IAA; 4) Method as described herein lacking the centrifugation step and lacking the step of stopping the reaction by addition on IAA and lacking the dilution step

[0174] FIG. 4 Comparison of the method as reported herein (1) with the methods as described in Matsumoto et al. ((2) GOD(GOx) activity assay; (3) HRP activity assay) with respect to activity, standard deviation and background.

EXAMPLE 1

[0175] High Throughput Screening of Sortase Activity from Crude Lysate

[0176] Cells (E.coli BL21) expressing sortase are cultured in 96 well plates with 200 μL LB-media.

[0177] Each cell suspension is lysed with lysis buffer (50 mM Tris pH 7.5, 0,1% TritonX-100, 200 mM NaCl and 5% B-PER) mixing it 1:10 and incubated at 50° C. for 30 min.

[0178] 50 μL of the lysate are mixed with 150 μL substrate solution (50 mM Tris pH 7.5, 200 mM NaCl, 20 μM of a glucose dehydrogenase (containing one of the substrates of the sortase reaction (LPXTG; here: X=K)) and 20 μM biotin (containing the other substrate of the sortase reaction (here: GGG))) and incubated for 2h at 37° C.

[0179] The reaction is stopped by addition of a 10- to 20-fold excess of inhibition buffer (50 μM Tris, pH 7.5, 200 μM NaCl, 10 mM CaCl.sub.2, 5 mM iodoa.cetamide). The stopped reaction mixture is centrifuged for 10 min at 5000×g.

[0180] The supernatant (50 μL) is transferred to a streptavidin coated multiwell plate and 100 μL of 50 mM Iris buffer (pH 7.5) comprising 200 mM NaCl, 10 mM CaCl.sub.2 is added and the mixture is incubated for 30 min at 30° C. at 200 rpm.

[0181] Thereafter the the multiwell plate is washed eight times with 300 μL washing buffer each (50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 5 mg/mL BSA, 0.1% Triton X-100).

[0182] After washing the multiwell plate 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L 4-nitrosoanilin, 1 mM CaCl2, 30 mM glucose) is added.

[0183] The kinetic of the reporter enzyme (glucose dehydrogenase) is measured over a time period of 5 min at 620 nm

[0184] The activity of the reporter enzyme is proportional to the amount of immobilized enzyme which is proportional to the amount of biotinylated enzyme and this is proportional to the activity of the sortase.

EXAMPLE 2

[0185] Measuring Sortase Activity

[0186] Purified sortase is mixed with its two substrates, the reporter enzyme, i.e. a glucose dehydrogenase containing the LPXTG (here: X=K) motif (60 μm) and a biotin derivative containing N-terminal glycines or alanines (60 μm) (1:1:1) in 50 mM Tris buffer pH 7.5 containing 200 mM NaCl.

[0187] This reaction mixture is incubated at 37° C. for 2h.

[0188] The reaction was stopped by addition of a 10- to 20-fold excess of inhibition buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 5 mM iodoacetamide).

[0189] The stopped reaction mixture is centrifuged for 10 min 5000×g.

[0190] The supernatant (50 μL) is added to 100 μL of 50 mM Tris buffer (pH 7.5) comprising 200 mM NaCl, 10 mM CaCl.sub.2 and streptavidin coted magnetic beads are added and incubated for 30 min at 30° C. at 200 rpm.

[0191] Thereafter the magnetic beads are washed five times with 300 μL washing buffer each (50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 5 mg/mL, BSA, 0.1% Triton X-100) in V-bottom multi-well plates using a magnet and a vacuum pump.

[0192] Afterwards the beads are resuspended in 100 μL citrate test buffer and 10-80 μL thereof are transferred to a new well. Thereto 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L4-nitrosoanilin, 1 mM CaCl.sub.2, 30 mM glucose) is added.

[0193] The kinetic of the reporter enzyme is measured over a time period of 5 min at 620 nm.

[0194] The activity of the reporter enzyme is proportional to the amount of immobilized enzyme, which is proportional to the amount of biotinylated enzyme and this is proportional to the activity of the sortase.

EXAMPLE 3

[0195] Measuring Sortase Activity with Different Method Setups

[0196] The experiments were basically performed as described above with some alterations:

[0197] Setup 1: method as described above (example 2)

[0198] Setup 2: like setup 1 but lacking the centrifugation step

[0199] Setup 3: like setup 2 but additionally also lacking the step of stopping the reaction by addition of IAA (iodoacetamid).

[0200] Setup 4: like setup 3 but additionally lacking the step of dilution step.

[0201] The substrate concentrations were chosen analog the publication of Matsumoto et al. (2011 and 2012) (5 μM Sort-tag LPKTG, 20 μM Nucleophile GGG-Biotin). The temperature, time of incubation and the enzyme concentration were chosen analog Matsumoto et al. (2012) (2,1 μM SrtA, 25C° and 30 min).

[0202] Results: (see FIG. 3)

TABLE-US-00002 Setup 1 Setup 2 Setup 3 Setup 4 Run A 0.3161 0.368 0.4539 0.5737 Run B 0.3318 0.3024 0.3281 0.7783 Run C 0.33 0.2856 0.2822 0.6901 Run D 0.329 0.2967 0.3915 0.5211 Run E 0.3272 0.3616 0.3901 0.8019 Run F 0.3152 0.3872 0.4083 0.4863 mean value 0.3249 0.3336 0.3757 0.6419 standard 0.0073 0.0436 0.0611 0.1342 deviation % variation 2% 13% 16% 21%

EXAMPLE 4

[0203] Measuring Sortase Activity, Standard Deviation and Background Signal Compared to Methods in Matsumoto et al. (2012)

[0204] The experiments were performed like described in example 2. The substrate concentrations were chosen analog the publication of Matsumoto et al. (2011 and 2012) (5 μM Sort-tag LPKTG, 20 μM Nucleophile GGG-Biotin). The temperature, time of incubation and the enzyme concentration were chosen analog to Matsumoto et al. (2012) (2,1 μM SrtA, 25C° and 30 min).

[0205] Data for GOx (termed GOD in Matsumoto et al.) activity and HRP activity was taken from Matsumoto et al. (2012).

[0206] Results for method as described herein (example 2):

TABLE-US-00003 Activity Background Run A 0.3161 −0.0002061 Run B 0.3318 0.0004713 Run C 0.33 −0.0002649 Run D 0.329 −4.93E−17 Run E 0.3272 0.0006773 Run F 0.3152 0.0002065 Mean value 0.3249 0.0001473 standard deviation 0.0073 0.0003757

[0207] Results compared to methods of Matsumoto et al. (see FIG. 4)

TABLE-US-00004 Method as reported GOx HRP herein (Matsumoto) (Matsumoto) Activity 0.3249 1200 0.26 standard deviation 0.0073 600 0.05 Background 3.6969E−05 200 0.12 Normalized 100%  100%  100%  Activity standard deviation 2% 50% 19% Background 0% 17% 46% Normalized on Activity