IPTG induction - no expression/induction (Jul/28/2005 )

hi all,
i've cloned my gene with his tag in pet28 and tried to express in BL21 DE3 pLys S, however i never see any induction with IPTG, at any concentration, time, temperature, this gene codes for a fusion protein of > 100 kDa and is from bacterial source, does anyone offer any clue what might be happening.
thanx
nbj

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There are many things that could have gone wrong. The first thing to check is if you still have the plasmid present in your cells.

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thanx,i'll definitely do plasmid stability test, but could u suggest me some more possibilities abt what might've gone wrong.
thanx again
nbj

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You can check IPTG with bacteria containing a plasmid that has an inducible lacZ gene and blue/white-screen them.

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hi there,
did you check to make syre your insert (gene) is inframe with the vector?

Jeff

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Hello nbj. Have you had any luck with your expression? I too am attempting to induce expression of an approximately 100 kDa protein in pET28 in BL21 (DE3) Star. The first few attempts yielded no expression. Induction under 25 and 37C. Overnight as well as 4 hours. IPTG at both 100uM and 200uM. The protein should be N-terminally tagged with polyhistidine meaning if there is any induction at all, there should be at least fragments of protein that should be detected by anti-6 his Ab. I haven't tried is checking the broth to see if the protein is being pumped out. We sequenced our construct and the his tag is still there and in-frame with the start. We could not confirm the sequence of the lac operator since we primed with T7 promoter.

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Check all first:
the existence of the plasmid;
the sequence of the clone;
IPTG;
host E.coli.
codon usage

If you're sure of all the things, try to change:

1. induction point : od600 =0.4~1.6
2 media: LB, TB, M9 etc
3. incubation time: 1hr - overnight
4. IPTG conc. : 10uM-1mM

I think, induction point is most critical for expression itself.
(LB. 1mM IPTG, 4hr incubation after induction)
if it does not work, change the media.


Good luck!!

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hi nbj,

have you ever tried that is there any protein expression without IPTG induction? I came across with a case where the protein that expressed out is toxic to bacterial cell growth. thus after adding the IPTG it inhibits the growth within 6 hours. You may see that the expression reduce as the time of IPTG induction increasing until zero.

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