Recombinant protein production
Plasmid expressing manose-binding protein (MBP)-tagged CCP21 or CCP22 domains of SVEP1, or MBP alone under the control of an iso-propyl-thio-β-glactosidase (IPTG) inducible promoter were transformed into E.Coli BCL21 cells. Transformed cells were grown in lysogeny broth (LB) media containing 100 µg mL-1ampicillin to an optical density of between 0.6 and 0.8 at 600 nm. Protein expression was induced by addition of 0.5 mM IPTG. Cell culture was pelleted, lysed and sonicated with the lysate cleared by centrifuging. The MBP-tagged proteins were immunoprecipitated from the cleared cell lysate using amylose beads (New England Biolabs). The protein was eluted from the beads using an affinity purification column with 10 mM maltose in PBS-T. The elution buffer was exchanged using spin columns with a MW cut-off of 30 KDa. Purified protein was run on a 4-12% Bis tris gel with protein visualised by Coomassie staining