I insert about 600bp cDNA of a gene in pET28a-His-SUMO plasmid, and trying to express this fused protein in BL21 STAR. But I can not induce this protein to express everytime. And I found a regulor pattern that when the strain grow up to A600=0.5 in 4 hours, it can not be induced but when the strain grow up to A600=0.5 over 7 hours, it can be induced. plz help me with this problem.
I always culture the strain in 5 mL LB overnight, and move 1mL to 50 mL new LB to culture , when the A600~0.5, I add IPTG to 0.2mM to induce
Hi,
I would try the induction at 0.6-0.8 with 1mM IPTG at 37C and 25C first before reducing the IPTG concentration to 0.2 mM or even more. Low concentration of IPTG works well for me always.
On a different note, Are you sure your gene of interest is inframe with Sumo? Have you done the restriction digestion to confirm the presence of your insert? it sounds like your insertion might have caused an early premature termination and that is why you can not see any induction of your target protein if I understand your problem correctly.
Hello Yupeng,
To me, it seems that you may have a problem with leakage of expression in the absence of IPTG, which results in the segregation of low or non producers. These have a faster growth rate than the high producers and will overgrow your precultures. Hencde the problem with your fast growing precultures. To avoid this, I would suggest a) that you generate a host strain with a tighter control by transforming it with a pLysS plasmid; b) that you make a fresh transformation of your recombinant pET28-His-SUMO into the new strain.
In addition to the suggestions above, you might also just try different temperatures. Sometimes 37oC is optimal, sometimes 30oC is, sometimes 23oC is.
- Badges
- Science topic