My 8 kDa protein cloned in pET21b+ expression vector not showing any expression. Protocol followsed for expression was pET21b+ clone transformed into a E.Coli BL-21 DE3 strain, Induced with 1mM IPTG and incubated at 37°C for 3 hour.
Buffer used for lysis was -
500mM NaCl, 20mM tris, 2mM Benzamidine and 10 µg/ml Soyabean Trypsin inhibitor.
Please suggest me ways to get expression of my 8 KDa protein.
To add to the suggestions above:
(1) I generally avoid induction at high temperatures as this tends to result in your protein localising to inclusion bodies / the protein becomes insoluble. I would recommend 20 degrees celsius.
(2) your lysis buffer isn't great - consider adding lysozyme to break open the bacterial cells, and preferably a detergent as well.
(3) protein induction is best *before* your bacteria reach the stationary phase; I usually induce around OD600nm = 0.6 (occasionally 0.8) with 0.1-0.5 mM IPTG at 20 degrees for a few hours by which point the OD is >1
(4) I would recommend doing a time course after induction - I once worked with a protein that was degraded by bacterial proteases 2 hours after induction (my protein triggered an induction of bacterial proteases, which then degraded it!), so I could only induce for 1 hour to see my protein! As an other extreme - some proteins are uber stabile and so overnight inductions are possible - this will give you more protein overall. Since your protein is very small, it's likely to be pretty soluble, so you might be able to get away with an overnight induction
(5) In your test experiments, do the following: lyse an aliquot of the induced cells directly in SDS-PAGE sample buffer (laemmli buffer) and run it out in addition to an aliquot lysed in your buffer of choice. This will give you an idea of whether your protein in expressed at all in the first place, and if yes, then whether it is soluble in your buffer.
Good luck!
After getting your recombinant strain:
1 - fermentation up to 2 units of OD-600 in flask scale typically in LB-medium
2 - induction at OD=1.5-2.0, post induction time 4hrs as a minimum - typically up to 6-10hrs (post induction time less than 4hrs is only for enzyme expression)
3 - your expression might be in form of IBs that requires refolding step
4 - your lysis buffer does not have the enzyme to do cell rupture therefore even if you have expression you cannot get it because no cell rupture
Hi Mohd,
First of all: There are a lot of mistakes you can make during expression and purification of your protein of interest, e.g. during expression (IPTG amount, induction time, temperature etc.) or cell lysis (method, buffer, native or denatured conditions). You have to check systematically if adaption of any of these parameters can improve your expression and protein yield.
You will find a very good guideline in the handbook “The QiaExpressionist”, available for free download.
Concerning the small size of your protein, it is possible that it is degraded during production in your host system. Small peptides are often fused to a DHFR protein for stabilization (see manual for further guideline).
All the best and good luck!
Ingmar
To add to the suggestions above:
(1) I generally avoid induction at high temperatures as this tends to result in your protein localising to inclusion bodies / the protein becomes insoluble. I would recommend 20 degrees celsius.
(2) your lysis buffer isn't great - consider adding lysozyme to break open the bacterial cells, and preferably a detergent as well.
(3) protein induction is best *before* your bacteria reach the stationary phase; I usually induce around OD600nm = 0.6 (occasionally 0.8) with 0.1-0.5 mM IPTG at 20 degrees for a few hours by which point the OD is >1
(4) I would recommend doing a time course after induction - I once worked with a protein that was degraded by bacterial proteases 2 hours after induction (my protein triggered an induction of bacterial proteases, which then degraded it!), so I could only induce for 1 hour to see my protein! As an other extreme - some proteins are uber stabile and so overnight inductions are possible - this will give you more protein overall. Since your protein is very small, it's likely to be pretty soluble, so you might be able to get away with an overnight induction
(5) In your test experiments, do the following: lyse an aliquot of the induced cells directly in SDS-PAGE sample buffer (laemmli buffer) and run it out in addition to an aliquot lysed in your buffer of choice. This will give you an idea of whether your protein in expressed at all in the first place, and if yes, then whether it is soluble in your buffer.
Good luck!
You may not see expression because of the low abundance of your protein. Collect a non-induced and a post-induction sample of your expression strain and analyze total and soluble lysates on SDS-PAGE revealed by western blot with anti-His6 antibody.
With the information at hand, I would say that you should first make sure that your protein is expressed at all in your experimental conditions following addition of IPTG, as suggested by Julien and Vikram;
check also if the sequence of your construct is ok and that the open reading frame is correctly positionned in regards to the translation start site.
What's the organism origin of your protein of interest?
@François-Xavier Campbell-Valois My Protein of interest is human origin.
@Julien B Henri I have load both pellet and supernatant from induced and uninduced culture respectively. but I didn't see any ~8 Kda Band in Induced Pellet Or Supernatant compared to uninduced one.
Make 2 constructs:
1) T7pro-Histag-GFP (control for induction + purification)
2)T7-HisTag-GFP-fused to ORF of your small protein (check on SDS-PAGE)
Check codons in your protein if you see rare codons like AGA or AGG, change them. Synthesize a new gene which fits E. coli translation system.
Insert a cleavage site of your choice between GFP and the protein of interest.
How did you conclude that it was not expressed? Was the expression plasmid confirmed by sequence?
Yeah Oleksiy Kovtun is right, before proceeding you must sequence the plasmid. Once the sequence is confirmed after then you should focus on other steps as mentioned above.
Please go up with the percentage of the gel you are using, could be it (or the degradants) migrates with the front, thus you simply don't see it...
best-jan
@Oleksiy Kovtun @Raihana Maqbool yes we had sequenced the expression plasmid and there is no mutation.
@Jan Piwowarski i had also checked it with Tris Tricine SDS PAGE and also with higher percentage of gel but didn't see any band ~ 8 KDa.
first I think your induced time is short,in our lab,we usualy induced for 6-8 hours,for your culture condition, the tempeture is another issue.when the constructed strain is needed to induced by IPTG,it must be at low tempeture,such as 30℃
Sorry for the next obvious concern... Have you seen it at any step expressed in or total, or inclusion bodies fraction? In your lysis buffer there is a high salt concentration can precypitate your protein, tus it is possible you have it in the pelet.
regards - jan
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