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Domains and fusions
 

Cloning or expression problems verses full-length, domains, and fusions

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 42C

 
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
15C
overnight
20C
overnight
25C
overnight
30C
5-6 h
37C
3-4 h
bulletHarvesting of the cell pellet by centrifugation (20 min at 6000 g). Cell pellets are stored at -20C.

 

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