Identification of a gluconic acid derivative attached to the N-terminus of histidine-tagged proteins expressed in bacteria.

Our previous studies have shown that the His tag cleaved from fusion proteins contained two distinct components P1 and P2. P1 has been identified to be a His-tagged peptide of G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R resulted from initiator methionine deletion, and P2 contains an unknown moiety at the second residue glycine of the tag (x-G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R, x = 178.0 Da). This study aimed to determine the structure of the modification by using a combination of protein isotope labeling and mass spectrometry. His-tagged FKBP was expressed in (15)N and (13)C labeling growth media respectively. Isotopic labeled His-tagged proteins ((15)N-His-FKBP and (13)C-His-FKBP) were isolated by affinity chromatography and subjected to Xa digestions to release the labeled His tag. Subsequent analyses of the released His tag by MALDI-TOF-MS indicated a mass difference of 178.0 +/- 0.2 Da, between the two (15)N-labeled peptides P1 and P2, suggesting that the modification moiety contained no nitrogen. A mass difference of 184.0 +/- 0.2 Da was observed on MALDI between (13)C-labeled peptide P1 and P2, indicating six carbons in the modification group. Also, comparing the mass shift on MALDI spectra of P1 and P2 after hydrogen/deuterium exchange revealed that the modification moiety had five hydroxyl groups. It was concluded that the modification was a gluconic acid derivative attached to the N-terminus of His-tagged proteins expressed in bacteria. The proposed structure was further confirmed by MALDI analysis of periodate oxidation products of His-tagged peptides.

Mass spectrometric determination of a novel modification of the N-terminus of histidine-tagged proteins expressed in bacteria.

Two proteins, FKBP, and Spo0F, were expressed in bacteria as histidine-tagged fusion proteins and isolated under native conditions. MALDI-TOF-MS analysis revealed that each protein preparation contained two components, neither of which corresponded to the molecular weights predicted from DNA sequences. The difference in molecular weight between the two FKBP components and two Spo0F components was approximately 178 +/- 14 Da. Site-specific proteolytic cleavage resulted in the release of histidine-tagged peptide from the recombinant proteins. MALDI mass spectra of the cleaved proteins showed a single molecular ion peak for each species with the predicted molecular weights. The histidine-tagged peptide released from both fusion proteins displayed two distinct peaks by MALDI-FT-MS corresponding to monoisotopic molecular weights of 2269. 027 Da and 2447.087 Da, respectively, which were both inconsistent with the predicted peptide sequence M-G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R of 2400.055 Da. The peptide at 2269.027 Da was sequenced by ESI-MS-MS and found to be a truncated histidine tag resulting from an initiator methionine deletion. ESI-MS-MS analysis of the peptide at 2447.087 Da indicated a moiety of 178.0 Da attached to the second residue glycine of the histidine tag. This alteration of the N-terminus does not fit any known modifications. A synthetic peptide with the identical sequence of the isolated his-tag M-G-H-H-H-H-H-H-H-H-H-H remained unmodified during the protein purification process, suggesting that modification of the initiator methionine was carried out in vivo, rather than the result of a chemical reaction from the isolation procedure.

Investigations for anthelmintic resistance in gastrointestinal nematodes from goats.

Field results from a commercial goat herd, based on egg counts and larval differentiation, suggested that anthelmintic resistance was present to albendazole, fenbendazole, levamisole, morantel, naphthalophos and phenothiazine. When the isolate was tested in sheep, using levamisole or oxfendazole, a possible resistance was shown for Trichostrongylus sp but no resistance was demonstrated in Haemonchus or Ostertagia spp. Ivermectin at a dose rate of 100 micrograms/kg was more than 98 per cent efficient in removing the adults of each of the three species of nematode. The phenomenon relating to the influence of the host on anthelmintic resistance is discussed.

Changes in response of a benzimidazole resistant strain of Haemonchus contortus from sheep after passing through calves.

A benzimidazole resistant strain of Haemonchus contortus was passaged through lambs only or from lambs to calves and back into lambs. Changes in response to thiabendazole were monitored by using an egg hatch test at each animal passage and by a controlled experiment on adult worms at the final passage in lambs. An increased level of resistance was shown for the isolate during its passage through calves by the egg hatch test, although this was not supported on the adult worms in sheep using a single dose rate of 66 mg/kg of thiabendazole.

Five generations of selection with benzimidazole and non-benzimidazole anthelmintics against benzimidazole resistant strains of Haemonchus and Ostertagia spp in sheep.

Benzimidazole resistant strains of Haemonchus contortus and Ostertagia spp were subjected to selection pressure over five laboratory generations with the recommended dose rates of either cambendazole, oxfendazole or morantel. A change in response, with larger residual worm burdens remaining after treatment at the fifth generation, was shown for both cambendazole and oxfendazole against both species of nematode. No change in response against either species are shown for morantel. The results are discussed in terms of the problem associated with inefficient removal of the adult parasites after treatment.

Patterns of wool moisture in merino sheep and its associated with blowfly strike.

A method for determining the moisture content of wool with a minimum disturbance to the constituents is described. The method was used to show differences in moisture content according to anatomical site, age, sex, strain of merino and in lesions of fleece rot, bacterial stain and active flystrike. The incidence of natural flystrike was recorded and related to the higher levels of moisture content. The moisture content in proximal wool was found to increase at different rates, following wetting of the fleece with droplets of water falling under gravity to simulate rain. A level of 20 per cent moisture content was reached after different time intervals in different strains of merino sheep, which may explain one of the differences in strain susceptibility to fleece rot

The effect of a drying agent (B26) on wool moisture and blowfly strike.

A drying agent (B26) was applied as a mist to sheep fleece. A reduction of 30 per cent in wool moisture was recorded, which persisted for 10 to 12 weeks. Reapplication of B26 extended the period of dryness, without adding to the reduction of moisture. Treatment using 100 to 200 ml of the drying agent resulted in reduced fleece moisture content for seven days when exposed to wetting by simulated rain. Treated sheep showed a 60 per cent reduction in the number and severity of fleece rot lesions. Diazinon reduced the incidence of natural flystrike by 50 per cent and the drying agent by 75 per cent over a five week period before shearing compared with untreated animals.