I normally pellet 1 ml of the expression culture to check expression. I then use a detergent lysozyme lysis method which works on our positive control. Other than errors in cloning, is there any other explanation?
Hi, Nandita!
Have you observed normal expression in previous experiments? If it's first time you are trying to express your protein, you can make some modifications:
1) prepare fresh inductor solution;
2) try to vary temperature;
3) try different concentrations of inductor;
4) change expression system
etc.
Also, if you can provide more details, community could provide you more pointed suggestions...
Good luck!
could be the condition is not suitable for your E.coli to express the protein or problem with your host, you could try other microorganism as host, e.g yeast
you can try and error to troubleshoot as you did not give much details on your problem
Firstly, Is your protein to be expressed from eukaryotes? Then there maybe odd codons when express in E.coli. Try some other restrains(Rosetta or Bl21 codon plus, et al).
If it is not the problem mentioned above, lowering temperature can increase the soluble protein and decrease the protein in inclusion body.
Some proteins indeed have low expression in E.coli. You can add some fusion tag which may augment the expression, like GST tag, GB1, MBP, also N termial His tag......
Hi,
Thanks for the responses. It seems the protein was expressed in the insoluble fraction which we managed to recover an a recent occasion
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