Purification and Characterization of Recombinant SP-A

NT-rfhSP-A was solubilised in 5 mM CaCl₂ and 5% glycerol (v/v), 8 M urea, pH 7.4 (solubilisation buffer) at 4 °C, overnight with mixing. NT-rfhSP-A was refolded by dialysis at 4 °C for 2 h against solubilisation buffer but with decreasing concentrations of urea (4 M, 2 M, 1 M and 0 M). After removal of precipitate, NT-rfhSP-A was purified using an IMAC purification column and cleaved through incubation with 10 units of thrombin (GE Healthcare) per mg of protein for 6 h at room temperature. rfhSP-A was purified by reapplication to an IMAC column to remove His-tagged NT. NT-rfhSP-A and rfhSP-A were analysed by SDS-PAGE under reducing conditions with subsequent Coomassie staining or analysis by Western blotting using a monoclonal mouse IgG antibody raised against nhSP-A. rfhSP-A identity and purity was confirmed by mass spectrometry using previously described methods (Simon et al., 2016 (link)).

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Watson A., Kronqvist N., Spalluto C.M., Griffiths M., Staples K.J., Wilkinson T., Holmskov U., Sorensen G.L., Rising A., Johansson J., Madsen J, & Clark H. (2017). Novel expression of a functional trimeric fragment of human SP-A with efficacy in neutralisation of RSV. Immunobiology, 222(2), 111-118.

Publication 2017

thrombin

Buffer Dialysis Glycerol Igg antibody Imac Mass spectrometry Monoclonal antibody Mouse Protein Reapplication Sds page Thrombin Urea Western blotting analysis

Corresponding Organization : NIHR Southampton Respiratory Biomedical Research Unit

Other organizations : Royal Brompton Hospital, Imperial College London, University of Southern Denmark, Swedish University of Agricultural Sciences

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Variable analysis

independent variables

Solubilisation of NT-rfhSP-A in 5 mM CaCl2 and 5% glycerol (v/v), 8 M urea, pH 7.4 (solubilisation buffer) at 4 °C, overnight with mixing
Refolding of NT-rfhSP-A by dialysis at 4 °C for 2 h against solubilisation buffer but with decreasing concentrations of urea (4 M, 2 M, 1 M and 0 M)
Incubation of NT-rfhSP-A with 10 units of thrombin (GE Healthcare) per mg of protein for 6 h at room temperature

dependent variables

Solubility and refolding of NT-rfhSP-A
Purification of rfhSP-A using IMAC columns
Analysis of NT-rfhSP-A and rfhSP-A by SDS-PAGE and Western blotting
Confirmation of rfhSP-A identity and purity by mass spectrometry

control variables

PH 7.4 of solubilisation buffer
Temperature (4 °C for solubilisation and refolding, room temperature for thrombin cleavage)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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