Characterisation of MBP-ZP-N-6his. (A) Schematic representation of MBP-ZP-N-6his fusion protein. (B) Multimerisation of MBP-ZP-N-6his. Purified protein, separated by SDS-PAGE under both reducing (R, lanes 2, 4, 6 and 7) and non-reducing (NR, lanes 3 and 5) conditions, was visualised by Coomassie staining (lanes 2, 3) and by immunoblot analysis with monoclonal anti-6his (lanes 4–7). Lane 1, Mr markers; lanes 6 and 7, progressively long exposures of lane 4. The position of bands corresponding to monomeric, dimeric and tetrameric MBP-ZP-N-6his is indicated. (C, D) Disulfide linkages of monomeric MBP-ZP-N-6his. The fusion protein contains 4 Cys residues, all within the ZP-N sequence. Native 1–4, 2–3 disulfides were assigned on the basis of MALDI-TOF-MS measurements of trypsin-digested MBP-ZP-N-6his, performed under non-reducing (NR, C) and reducing (R, D) conditions (Methods). MBP and ZP3 amino acid numbers refer to database entries 1HSJ_A and P10761, respectively. Peaks represent average mass/charge ratio (m/z). Disulfide-bonded and free Cys-residue containing peptides are marked by blue and red circles, respectively; LEH6 C-terminal tag peptide is marked by a black circle; peaks with intensity below 5% are indicated by dashed circles.