My cloned gene in pET24a contains C-terminal His tag and Kan resistance. it's not getting expressed in Origami cells and BL21cells, and my transformation was good (in Both Cells).Anyone have any suggestions for troubleshooting this experiment. Protein Molecular weight is 66 kDa. Using 1mM IPTG Concentration.
1. How do you know it isn't being expressed? Could it just be expressed at a lower level? Is it getting cleaved by a protease? Could it be insolube and precipitating?
2. Silly question, but are you using the versions of Origami and BL21 with T7 polymerase - Origami (DE3) and BL21 (DE3)? Ideally you should compare, for example, BL21 to BL21(DE3) so you can see expression with and without T7 polymerase.
3. What OD do you grow to before induction? Have you tried different IPTG concentrations? Is your IPTG old?
4. Maybe double check the N-methionine excision (NME) to see if your methionine is likely to be lost and the N-end rule (NER) to see if your protein might get degraded if the second amino acid becomes the first.
I'm sure I can think of more if that doesn't work.
1. How do you know it isn't being expressed? Could it just be expressed at a lower level? Is it getting cleaved by a protease? Could it be insolube and precipitating?
2. Silly question, but are you using the versions of Origami and BL21 with T7 polymerase - Origami (DE3) and BL21 (DE3)? Ideally you should compare, for example, BL21 to BL21(DE3) so you can see expression with and without T7 polymerase.
3. What OD do you grow to before induction? Have you tried different IPTG concentrations? Is your IPTG old?
4. Maybe double check the N-methionine excision (NME) to see if your methionine is likely to be lost and the N-end rule (NER) to see if your protein might get degraded if the second amino acid becomes the first.
I'm sure I can think of more if that doesn't work.
Hello Mam, Firstly I would like to thank you for giving me a suggestion.
1.Bcoz its mine third time to check protein expression, particular for this protein that I was asking. It's not getting cleaved by a protease and not insoluble.
2.its an Origami2 (DE3) and, BL21(DE3).
3.i am using OD before induction is .6-.8. yes mine IPTG was old (i think some problem was there with old IPTG)
I will follow your suggestion.
Hi Shivam,
When the protein of interest cannot be detected through a sensitive technique (e.g., Westernblot) or it is detected but at very low levels (less than micrograms per liter of culture), the problem often lies in a harmful effect that the heterologous protein exerts on the cell. So to solve protein toxicity problem, use promoters with tighter regulation and lower plasmid copy number and pLysS/pLysE bearing host strains in T7-based systems or use strains that are better for the expression of toxic proteins (C41 or C43). You may also need to start induction at high OD, shorten induction time, add glucose when using expression vectors containing lac-based promoters, use defined media with glucose as source of carbon.
Good luck
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