E.coli Culture

Culture stock E.coli containing a plasmid. Add 1ml of the glycerol stock in 200 ml of fresh LB/kanamycin medium. This vector is pUC which is a high copy plasmid for cloning. For better protein expression, 180 rpm shaking and a lower culture temperature such as 25C reduces copying plasmid and increases expression of recombinant protein. It may take a longer incubation period to reach OD600 at 1.0 – 2.0 (two days or longer). Once the OD reaches OD 1.0 – 2.0, expand the culture from 200 ml in 2 L (1.1 L each would work). Then, culture at the same condition overnight. Culture the cell until OD600 = 1.0 – 2.0.

Cell Harvesting

Centrifuge the cell suspension at 10,000 rpm for 15 minutes at 4C. Save pellets in a 50 ml centrifuge tube (pre-weight, empty) and weigh wet weight (Post-weight can be subtracted pre-weight to obtain pellet weight, generally 3g per 1L). The supernatant can be stored at 4C. The pellets (cells) can be stored at -80C until use.

Lysate Preparation

All procedures must be on ice all times.

Thaw the cells on ice and suspend the cells in Lysis Buffer below at 5ml per gram wet weight.

Lysis buffer

100 mM Tris·Cl, pH 7.0

300 mM NaCl

5 mM EDTA

5 mM DTT

5 mM benzamidine·HCl

Add egg white lysozyme at 1mg/ml and incubate on ice for 30 minutes with a mixing tube occasionally. Sonicate lysate with medium intensity at least 6, 10-second bursts with swirling in between each interval, or until lysate looks noticeably less viscous. Centrifuge the lysate down at 10,000 RPM for 15 minutes at 4C. The pellets must be saved at -80C for incase of insoluble (Inclusion-Body) recombinant proteins which require further solubilization. Make 1 ml aliquot for the analysis of the purification process, then store at –20C until use (1).

1. P. T. Wingfield, Preparation of Soluble Proteins from Escherichia coli. Curr. Protoc. Protein Sci. 78, 6.2.1–6.2.22 (2014).