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IPTG Induction of recombinant protein production



IPTG Induction of recombinant protein production

IPTG induction and bacterial growth
A introduction and experimental protocol on BL21 e.coli recombinant protein expression and induction with IPTG. (Structural biology Protein preparation protocols, South Africa Structural Biology Initiative)

The induction of recombinant protein expression is effected by addition of the non-metabolisable lac-inducer IPTG. Although the process is simple in theory, the BL21 cells tend to form inclusion bodies very easily. This reduces recovery dramatically and therefore in the past, each recombinant protein had to be assessed individually for their solubility and expression.

IPTG Induction and Extraction of Proteins from Bacteria (by Swathi Arur and Sudhir Nayak)

Induction in bacteria can be performed using one of two basic methods. Fast induction does not work for all proteins and can give you suboptimal yields. Slow induction can enhance the solubility of some proteins.

GST Fusion Protein Production through IPTG induction

This protocol uses pET Expression System and BL21(DE3) E. coli.

Last update 18-Sep-2006, Rating of 17 votes.

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By ggkk on 24-Feb-2009
i am expressing 130kd protein. using a qiagen expression vector. 1mmIPTG for 6 hours . reference strains are working but not my clone. checked on 12% resolving gel . what to do?
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By massimiliano on 02-Dec-2008
I use pET22b and IPTG 1mM 5 hours induction at culture Abs=0.9 37C
bye
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By Kátia Morais on 12-Nov-2008
I use BL21 with vector pDEST17 system from Invitroge. My construction expressed induced with L-arabinose, but in last experiments I didn't get expression of my protein. So I tried with IPTG and it worked. Does anyone here know how IPTG act about L-arabinose Operon or why L-arabinose didn't work (maybe instability)?
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By Ahmed on 10-Nov-2008
Has anyone found an optimal [IPTG] when using the pET23a vector for protein expression (~21kDa)?
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By Shivasankari on 05-Nov-2008
After addition of IPTG the multiplication of cells greatly lowers, this because most of the energy is shunted towards protein synthesis. So it is good when the no of cells in induced culture is greater than those of the uninduced. Optimum concentration of IPTG depends on the protein, vector, etc , so there is nothing universal in IPTG concentration , it has to be standardised using ones own hands. To start with 0.5 mM will be optimum and then go beyond or less than that, if at all there is induction. it is advisable to not go beyond 1mM IPTG.
At times the protein conc may be more in uninduced than induced, this is because of leaky expression. And the same would happen in the induced also, but since the no of cells is more in uninduced than induced, you get an illusion that more protein is in uninduced than induced. To rule out this try to take equal no of cells from induced and uninduced - this can be done by taking cells based on O.D. .. Hope this helps...

Alas I am also struggling to get my protein which is in pET 15b ... I am not even able to see my band in the gel...


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By irum nawaz awan on 28-May-2008
i am also expressing my gene in Bl21DE3pLysS strain as i didn't get expression of my protein in Bl21DE strain.Whats the reason that in some cases our protein expression is equal or more in uninduced than induced ones seen in SDS-PAGE?
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By chandra on 15-Mar-2008
hi..i m also doin' the same xprmt using BL21 star w' pET, but pET101/D-TOPO. since your SDS doesn't shows any results, hv you tried the western blottin' for detection? if there are no detections, might be your ligation to the vector didnt work.
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By N.PADDA on 19-Feb-2008
Iam facing troubles with assay of NAG N-acetyl D-glucosamine(Reisseig et al,1954) .activity is observed on plate but not detected in assay.
Can anyone please suggest me its solution?
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By jhansi on 03-Jan-2008
i use BL21 with vector PET system from novagen.i tried different conentrations of IPTG.By running SDS gel,it does not show my protein.
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By ARUN on 31-Oct-2007
what will happen if we induce the cells with IPTG before 0.4 OD?(to be precise at 0.37)Will there be any variation in the amount of the protein expressed? If induced before 0.4 OD then do we need to increase the incubation time of the culture in the shaker? thanks in advance.
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By siddhu on 18-Oct-2007
IPTG acts by lowering the problems of inclusion bodies and you can get soluble protien .normally induction is done when the cells are growing at mid log phase from 0.5 to 0.7.
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By alkprotease on 12-Oct-2007
IPTG with higher conc. will inhibit the growth and u plz tell me the conc. of IPTG u used and time of incubation after induction.
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By Nus on 27-Sep-2007
I trying to do protein (19kDa) expression by using the pet19b vector. Surprinsingly I detected a stronger expression without IPTG than with IPTG.
Does anyone know the reason?
Thanks!
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By S.M. on 02-Mar-2007
Has anyone found an optimal [IPTG] when using the pET15b vector for small protein expression (~4kDa)?
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By sanju on 19-Feb-2007
i am doing protein expression by using IPTG.
how it will act in expression?
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By robinson on 28-Nov-2006
have you taken care that you sonicated or lysed the cell after an incubation of 6 hours after inducing with IPTG. measure the OD before lysing. I found better yield after an od of 1.4 which may take around eight hours if you induced at 0.4 OD
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By lt on 18-Sep-2006
I use same cell BL21 with vector pNET system from Novagen. I add IPTG after OD 0.4-0.6. What happened is cells with IPTG growing much lower than negative control ones (without IPTG). By running a SDS-Page gel, it show my gene did not have expression on this experiment.
Dose anyone know the reason?
My Gene has GC rich region at begining.
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