Functional Assays

In dot blots the anti-α-LTX antibody (ALOMONE) reacted to 20 nM α-LTX at a dilution of 1 : 100 but not at a dilution of 1 : 500. This low sensitivity was confirmed when 60 ng α-LTX (15 µl of 20 nM solution) was run as a protein sample on an SDS-gel and analyzed in an immunoblot. Only a weak band appeared in the blot with the antibody diluted 1 : 500, but at a dilution of 1 : 100, the reaction seemed to be strong enough to detect lower amounts of α-LTX (data not shown). Another anti-α-LTX antibody from a different supplier (SIGMA) failed to react with α-LTX.

The detection of α-LTX by anti-α-LTX antibody in binding assays with N-termini of C. oncophora and O. ostertagi depsiphilin within FPLC eluates (described in chapter 5.24.2) revealed three very weak bands, approx. 55 kDa, 45 kDa, and 36 kDa in size.

The largest and the smallest bands had the same size as the bands detected by the specific anti-Hc110-R antibody from project 8214; the middle band was different (see Figure 17). In control blots with 50 % glycerol without α-LTX, no bands were detected by the anti-α-LTX antibody. To test α-LTX for nonspecific binding to proteins, several other highly expressed recombinant proteins and O. ostertagi raw antigen were run in an α-LTX binding assay and tested for their binding capacities. Specific binding of α-LTX to depsiphilins could not be confirmed; moreover, the best binding partner for α-LTX in this experiment was N-methyl-transferase (NMT) of the bovine lungworm D. viviparus (see Figure 18). The stained band in lane 1 was not identical with the main depsiphilin band in the Coomassie-stained gel.

The On-Blot binding assays of α-LTX to refolded recombinant depsiphilin N-termini of C. oncophora and O. ostertagi can therefore not be regarded as specific binding.

128 Results

Figure 17: α-LTX blot of a preparative SDS gel with recombinant C. oncophora depsiphilin N-terminus (Co pDEST 17). Lane L: SeeBlue^® Pre-Stained Standard, lanes 1 – 3: blot incubated with α-LTX in 50 % glycerol, lanes 4 – 6: blot incubated in 50 % glycerol without α-LTX. Lane 1:

primary antibody anti-α-LTX antibody 1 : 500, lane 2: primary antibody anti-α-LTX antibody 1 : 100, lane 3 and 4: primary antibody Sample 4 rabbit 5062 1 : 2000, lane 5: primary antibody anti-α-LTX antibody 1 : 100, lane 6: primary antibody anti-α-LTX antibody 1 : 500. Secondary antibody anti-rabbit IgG (whole molecule) Alkaline Phosphatase conjugate 1 : 3000. Arrows mark the very weak bands in the lane with α-LTX and anti-α-LTX antibody

Figure 18: Coomassie-stained gel (left) and α-LTX blot (right) with recombinant O. ostertagi depsiphilin N-terminus (Oo pDEST 17) and other proteins. Primary antibody anti-α-LTX antibody 1 : 100, secondary antibody anti-rabbit IgG (whole molecule) Alkaline Phosphatase conjugate 1 : 3000. Lane L: SeeBlue^® Pre-Stained Standard, lane 1: Oo pDEST 17, lane 2: raw antigen (supernatant) of O. ostertagi adults, lane 3: raw antigen (pellet) of O. ostertagi adults, lane 4: raw antigen (pellet and supernatant) of O. ostertagi adults, lane 5:

major sperm protein of D. viviparus, lane 6: protein disulfide isomerase of D. viviparus, lane 7:

NMT of D. viviparus, lane 8: paramyosin of A. caninum. Arrows mark the stained bands in lane 1 (depsiphilin) and lane 7 (NMT)

kDa 98 kDa

98 64 64 50 50 36 36

L 1 2 3 4 5 6 7 8 L 1 2 3 4 5 6 7 8 98

64 50 36 kDa

250

L 1 2 3 4 5 6

Results 129

6.7.6.2 Dynabeads^®

α-LTX coated Dynabeads^® were also used for testing α-LTX for specific binding to the recombinant depsiphilin N-termini. In experiments with Dynabeads^® coated with the One-Step coating procedure, no α-LTX was detected after boiling the particles for elution. A band the size of depsiphilin’s N-terminus was found. These results indicate a nonspecific binding of recombinant protein to the beads and a failure of the coating procedure.

In experiments with particles coated with the Two-step coating procedure, two bands were present in the Coomassie-stained gel after loading the supernatant of boiled particles; one of approx. 130 kDa (α-LTX) and one of approx. 55 kDa. The pattern of bands in the immunoblot with the anti-Hc110-R antibody from project 8214 was identical to the pattern of the depsiphilin’s N-terminus, whereas the anti-α-LTX -antibody seemed to partially stain the depsiphilin as well (Figure 19).

Figure 19: Coomassie-stained gel (left) and immunoblot (right) of an experiment with Dynabeads^® coated with α–LTX with the Two-Step coating procedure. Target was purified recombinant O. ostertagi depsiphilin N-terminus. Lane L: SeeBlue^® Plus2 Pre-Stained Standard, lanes 1 and 2: boiled dynabeads (= eluate). Arrows indicate the expected sizes of α–LTX (130 kDa) and depsiphilin N-terminus (approx. 55 kDa). Immunoblot: Lane 1 blotted with specific anti-Hc110-R antibody sample 4 of rabbit 5062 (project 8214) 1 : 2000, presenting the typical pattern of three bands; lane 2 blotted with anti-α–LTX 1 : 100. Secondary antibody for both lanes anti-rabbit IgG (whole molecule) Alkaline Phosphatase conjugate 1 : 3000

98 64 50 36 kDa 250 kDa

250 98

36 50 64

L 1 L 2 L 1 L 2

130 Results

This result was tested for its specificity. One experiment compared the binding affinities of α–LTX coated particles to the depsiphilin N-terminus in presence of bovine serum albumin (BSA) and the binding to BSA alone without the depsiphilin N-terminus. These experiments showed a nonspecific binding of BSA to the beads and, moreover, a nonspecific binding of anti-α–LTX antibody to BSA (data not shown). In another experiment a sample of beads was processed with the Two-Step coating procedure but without α–LTX. The nonreacted activated carboxylic acid groups were quenched as previously described (5.24.3). These beads were incubated with depsiphilin N-terminus protein, and elution was performed by boiling the particles. The eluate contained the protein (Figure 20). Hence the recombinant N-terminus bound to the particles nonspecifically.

Figure 20: Immunoblot with samples of an experiment with Dynabeads^® coated without protein.

Primary antibody sample 4 of rabbit 5062 (project 8214) 1 : 2000, secondary antibody anti-rabbit IgG (whole molecule) Alkaline Phosphatase conjugate 1 : 3000. Lane L: SeeBlue^® Pre-Stained Standard, lane 1: target sample O. ostertagi depsiphilin N-terminus, lane 2:

supernatant after wash step with Tris buffer, lane 3: supernatant after wash step with PBS buffer, lane 4: beads loaded onto the gel after boiling (eluate). Arrow marks the size of the main depsiphilin band

98 64 50 36 kDa 250

L 1 2 3 4

Results 131

6.8 Eukaryotic Expression