The expressed product from the pRSET-MYOC plasmid produces a
fusion protein that adds 32 amino acids to the N terminus of MYOC
(Note: This replaced the 32 amino acid signal peptide). Within the 32
amino acids are a 6 amino acid histidine tag (for purification) and an
8 amino acid epitope that is recognized by monoclonal antibody
anti-Xpress (Invitrogen).
Plasmid pRSET-MYOC, pRSET-β-galactosidase or pRSET-B (empty
expression vector serving as negative control for purification) were
transformed into BL21(DE3)pLyS bacteria. Overnight cultures were
diluted to 0.1 (OD600) and allowed to grow until
OD600 reading was 0.4 to 0.5 (exponential
growth). The culture was allowed to grow for 3 additional hours, and
then cells were collected by centrifugation. For purification of MYOC,β
-galactosidase, and control proteins (pRSET-B), bacterial pellets
were lysed in 50 mM sodium phosphate buffer (per liter: 46.6 ml of 1 M
Na2HPO4, pH to 8.0 with 1 M
NaH2PO4), 300 mM NaCl, 10
mM imidizole, and 1 mg/ml lysozyme. Bacterial lysate was sonicated,
pulled through a 23-gauge needle, and centrifuged to remove debris.
Supernatant was collected and 1.5 ml of Ni-NTA agarose (Qiagen,
Valencia, CA) was added. The sample was allowed to rock at 4°C for 1
to 2 hours and centrifuged, and the supernatant was discarded. Ni-NTA
agarose was washed six times with 10 ml of 50 mM sodium phosphate
buffer, 300 mM NaCl, and 20 mM imidizole. Two additional washes were
performed with 50 mM sodium phosphate buffer, 300 mM NaCl, and 50 mM
imidizole. Protein was eluted from Ni-NTA agarose with 1 to 2 ml of 50
mM sodium phosphate buffer, 300 mM NaCl, and 250 mM imidizole. Isolated
protein was dialyzed overnight against buffer containing 50 mM sodium
phosphate buffer, 300 mM NaCl, and decreasing amounts of imidizole (100
mM → 50 mM → 0 mM).