Protein Expression and Purification Series - Bio-Rad

APPENDIX A
TIPS AND HELPFUL
HINTS
Protein Expression and Purification Series
Appendix A: Tips and Helpful Hints
How to get the best protein expression and purification
results
1. Culture, subculture, and induction
The time frames listed in the instruction manual are designed to have as much flexibility as possible to allow
for classroom time periods. However, when possible, the following guidelines will help you achieve the best
results for cell growth and protein expression.
• Initial overnight cultures should be between 14-20 hours rather than 24 hours.
• Induction levels are the highest when induction occurs for four hours.
The figure below shows a comparison of cells induced for four hours versus 24 hours, demonstrating the
clearer induction of the cells induced for four hours.
250 kD
150
100
75
50
37
25
20
15
10
1 2 3 4
Figure 1. Gel comparing four-hour induction to 24-hour induction.
The four-hour induction lane has a much more defined band of
GST-DHFR-His (lane 3, arrow) relative to all of the background E. coli
proteins when compared to 24 hours of induction (lane 4, arrow).
Lane Sample
1 Precision Plus Protein Dual Color standards
2 Uniduced cells
3 Cells induced 4 hours
4 Cells induced 24 hours
2. Stability of induced protein
While the BL21(DE3) E. coli are deficient in some proteases, extra protease inhibitors are not being added
to the cell lysate due to chemical safety.
• After induction of the BL21(DE3) cell culture, the best stopping point for protein stability is after
pelleting the cells and removing the supernatant to form what is called a cell paste.
• This cell paste is highly stable when stored at –20°C or –70°C.
3. Lysis of cells
The most efficient lysis of cells requires the cells to be completely resuspended followed by complete
freezing and thawing of the resuspended cells.
• Completely resuspend the cell paste in the lysis buffer containing lysozyme. This may require vortexing
and vigorous pipeting.
• An ethanol/ice bath is the preferred method of freezing.
• Thaw frozen lysed cells completely before beginning each subsequent freeze-thaw cycle.
• If an ethanol/dry ice bath cannot be used, the freeze-thaw can be accomplished in a –70°C freezer
overnight or by two cycles of overnight freezing followed by thawing using a –20°C freezer. A –20°C
210 Appendix A: Tips and Helpful Hints