| Domain or fusion |
Difficult cloning |
No or variable
expression |
Low or variable
yield |
Insoluble |
Unstable |
Dysfunctional |
| Full-length |
Most likely to have problems. |
Problems of toxicity and codon usage. |
Lowest yield. |
Likely if contains hydrophobic domains. |
May need ligand, cofactor, prosthetic group or
chaperones. |
May need ligand, cofactor, prosthetic group,
chaperones, or modifications. |
| Domains |
Occasionally have problems. |
Codon usage only.
Reduce toxicity significantly. |
Generally increased yield. |
Some domains may become soluble. |
May be helpful. |
May not be helpful. |
Large fusion
(GST,Trx and MBP etc.) |
May work because of reduced toxicity. |
Reduced toxicity and codon usage problems. |
Generally increased yield. |
May increase solubility. |
Increased stability in some cases. |
May not be helpful. |
Small fusion
(His-tag) |
Mostly same as intact protein. |
Mostly same as intact protein.
Slightly reduced codon usage problems. |
Mostly same as intact protein.
Reported increase yield in some cases. |
Mostly same as intact protein. |
No significant difference. |
May not be helpful. |
Different Choosing different strategies may solve some problems. Other
expression problems may still remain. In some cases, these cloning strategies may not make significant difference
in a chosen expression system. There is no single cloning strategy can solve all
protein expression problems. Multiple strategies may be chosen for an
expression. However it also increases time, labor, and cost significantly.
For the most used promoters induction conditions are listed below.
| Promoter |
induction |
typical condition |
range |
| trc (hybrid) |
addition of IPTG |
0.2 mM |
0.05 - 2.0 mM |
| araBAD |
addition of l-arabinose |
0.2% |
0.002 - 0.4 % |
| PL |
shifting the temperature |
from 37 to 42°C |
|
| T7-lac operator |
addition of IPTG |
0.2 mM |
0.05 -2.0 mM |
After induction the cultures are incubated from 3 hours to overnight
depending on the induction temperature. Guide lines are given below.
| Incubation temperature |
incubation time |
| 15°C |
overnight |
| 20°C |
overnight |
| 25°C |
overnight |
| 30°C |
5-6 h |
| 37°C |
3-4 h |
 | Harvesting of the cell pellet by centrifugation (20 min
at 6000 g). Cell pellets are stored at -20°C. |
Your comments and suggestions are greatly appreciated at
info@exptec.com.
Expression Technologies Inc. 
