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