I am working with a protease gene which is his-tagged at c-terminal and is ~50Kda size. I transformed the recombinant construct (pET23b+Gene) in Rosetta DE3 pLysS. When I am trying to check induction I am getting results like that n given SDS PAGE.
The conditions are:
Induction with 0.6mM IPTG when od reaches 0.6
Lane-2 marker (97.4kda, 66kda 43 kda 29kda 20.1 kda and 14 kda)
lane-3 uninduced cells 200ul
lane-4 induction of 2 hours
lane-5 induction for 4 hours
lane-6 induction for 6 hours
lane-7- blank
lane-8 20 hour induction.
As you can see the gel image expression is going on but its giving band of much lower size than expected. I have done sequencing/ primer walking of my gene, they are intact and 100% correct.
Hi there,
your protease is degraded. Have you tried to identified the content of the two bands by MS fingerprint?
Dear Vishal
Your expected protein is protease and its induction may be induce proteolysis.
This can produce several small size bands which are not related to your experiment.
It is better to use protease inhibitor plus your samples
' am planning. To do mass finger print.. if I will add protease inhibitor then my protein will also not work
Your protease could either be degrading itself (autoproteolysis), or be being degraded by an E. coli protease. A protease inhibitor could prevent both, but would add to the cost. Purification of the protease and dialysis will remove the inhibitor. What temperature are you doing the induction at? I would try to force the protease into inclusion bodies by inducing at 37oC for 2 hr, then you would have to purify under denaturing conditions, and then refold it.
Gary i am dong induction at 37 degree and induced it from 2-20 hours as shown in gel. so only option is to add inhibitors.
In your 20h induction sample you have a 2nd band with a higher molecular weight. This band is absent or very faint in your other samples. Is it possible that this is your protease?
Vishal,
I would do a time course after induction (in minutes: 2.5, 5, 15, 30, 60, 90) in order to confirm that the full-length protein does accumulate at an early time point. If that is the case, then you could do a shorter expression time at 37oC with more cells to get enough protein. You could also try expression at 15oC and 24oC-for longer time points. Since the protein is soluble, expression at 37oC will only enhance the breakdown of the protein by either mechanism, while lower temp. expression may help the full-length protein to accumulate.
And as pointed out by Max, the 20 hr time-point has two bands, the MW of each when added together appears to add up to ~50 kDa. Maybe an E. coli protease is degrading your protein into two main protein bands, and then further degrading the larger band into peptides. But after 20 hr the E. coli protease has lost most of its activity, and not replaced by fresh enzyme, resulting in the larger band accumulating.
Svi fenomeni u prirodi imaju svoju empirijsku i misaonu stvarnost. Suštinu date empirijske stvarnosti možemo spoznati uz pomoć teorije sistema i kibernetike. Kada spoznamo suštinu stvarnosti dobit ćemo odgovore na sva pitanja.
Hi Vishal,
Certainly add protease inhibitor cocktails as everyone has suggested but also try reducing induction temperatures and/or try auto-induction. These conditions may improve read-through of your protein during translation and prevent 'stalling' when you have a string of low usage codons for example. Stalling is frequently seen with e.g. GST fusions where you see lots of the fusion partner but your fusion protein is incomplete, for some proteins you may even see a 'ladder' of incomplete translates. 37°C induction with IPTG pushes everything along really hard and E.coli ribosomes just 'give up' on a protein when they hit a problem (low usage codons, slow folding of protein etc.) so slowing the process down can dramatically improve the amount of full length protein you produce.
Is there any documentation to suggest autolysis of your protein? Again the inhibitors may help but if it is a long term you may want to think about mutating out this function.
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