Express the Maltose Binding Protein (MBP) in ER2566 E.coli with the pMAL-pIII plasmid.
- The E.coli cultures grown overnight were centrifuged at 4500rpm for 15 minutes at 4°C.
- The supernatent was discarded and the cell pellets were each resuspended in 1mL of rich broth (4mL total).
- The resuspended pellets were evenly divided into four 2800mL flasks that contained 1L of rich broth with 100μg/mL ampicillin.
- These flasks were then shook at 165rpm at 37°C for about three hours. At this point 1mL of 0.1M IPTG was added to each flask.
- The cells continued to grow and now express protein for about three more hours at 165rpm at 37°C.
- The cultures were centrifuged at 4500rpm for 15 minutes at 4°C. In total the four pellets were resuspended in 200mL of column buffer. The cultures were then stored at -20°C.
- Column Buffer: 20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA