I work on eukaryotic gene, I want to clone and express it, I transform my recombinant plasmid to Ecoli(BL21), after induction, my protein does not express. I don't see my protein band in SDS electrophoresis
Some recomendations.
1. Always check the sequence of the expression construct.
2. Use freshly transformed cells to check expression
3. Use a positive control
4. Check expression in total lysates and the soluble and insoluble fractions
Hello Prastou
Do you have a positive control that you can run in. If you can not see your protein expression and getting expression for the positive results, this means that your operon is staying closed in the conditions where you are performing expression. My recommendation is to change the conditions of expression like temperature, induction time... If you can give more details about expression, we can make better thinkings, like your plasmid, temperature, amount of rare codon of the protein, IPTG concentration.
I hope you will express it.
Good Luck
Some recomendations.
1. Always check the sequence of the expression construct.
2. Use freshly transformed cells to check expression
3. Use a positive control
4. Check expression in total lysates and the soluble and insoluble fractions
good recommendations.
What do you mean by SDS PAGE... Coomassie stain? Western Blot? Silver stain? If you have your protein tagged, or you have an antibody, please try Western blot, so you know what you are looking at. It's not uncommon to get decent expression, but you don't see it in the background of other E.coli proteins. Western is more specific.
And the bottomline from comments earlier, I would say:
1.After you've verified your material,
2.Optimize your protein induction with the suggested variables, most importantly, induction time and temperature. Use small cultures, Check the results of all your tests by doing a crude protein extraction (=soluble + insoluble) by pelleting bacteria (~equivalent to 0.5ml OD1), and boiling pellet 5min in ~50ul SDS protein sample/loading buffer, then spin and load some of the sup on gel. And do a Western if you can.
3. Optimizing solubility and extraction etc.. you can do after that..
Check these things first, if nothing works, you need to go back to the drawing board and worry about codon usage, toxicity, protein cleavage etc. But see what you've got first.
1. Where did you get your DNA from?
PCR from genomic DNA ( hen you still have your introns in the sequence) which is a problem.
From mRNA, did RT step work correctly?
Synthesized DNA insert?
Did you sequence your DNA, with promoter and terminator? Is there a Shine-Dalgarno sequence, maybe point mutations in P or T?
2. Expression
Did you try different IPTG (or other inducers) concentrations?
Try different expression temperatures 20-37 degrees
Expression with AB?
Grow cells to OD 0.3-0.5 then induce
Is your gene toxic for the cells? for example cell wall digesting proteins or something like that, expression of these proteins can kill the cells.
Do you maybe have inclusion bodies? Try to dissolve them with 8M urea.
3. SDS-PAGE
whats the size of your protein, maybe too small proteins gets degradet or cleaved by proteases after lyses of the cells. Use protease inhibitor (e.g. ROCHE)
Did you try to blot your gels? and detect your protein?
Once you are through with all preliminary protein expression check as mentioned in the above answers and still fail to get the expression, then there is a chance that your protein might be toxic to the cell. You can switch over to the vector host systems which gives tight regulation of gene expression (pBAD vector). Alternatively, you could try E coli BL21DE3 pLys strain.
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