his-tag protein pure but degradated during growth - his-tag protein pure but degradated during growth (Apr/28/2008 )
QUOTE (matys @ May 5 2008, 10:41 AM)
BL21 recombinant cells are grown util a A600=0.8-1.0 at 30°C, then the culture is shifted at 20°C and induced with IPTG 1mM for 1 hour. Cells from 1 liter of culture are collected and directly resuspended in 50ml of 20mM sodium phosphate buffer pH 7.4, 0.5M NaCl2, UREA 6M, DTT 3mM, Imidazole 30mM and Protease EDTA-free inhibitor cocktail 1X (1 tablet). Cells are disrupted by sonication. After centrifugation, the lisate is loaded onto a nichel-sepharose column equilibrated with ther lysis buffer (without inhibitor cocktail). After washing with the same buffer, the protein is eluted with 250mM imidazole added to the same buffer. What do you think about it?
It sounds like a good protocol
but maybe you should take a look at the test that I propose you 
-Jipes-
Yes, I'm going to try the test you've suggested, and I'll let you know the results!
-matys-
I've just obtained the results of the test proposed by Jipes.
I've prepared 50ml of recombinant cell colture, let it grow at 30°C and when it has reached OD 0.8 I've shifted the growth temperature at 22°C. I've collect 1ml of the culture each 30 minutes up to 3 hours, and I've resuspended each pellet directly in sample buffer. Finally I've loaded all samples in sds-page, trasferred the gel on NC and done the incubation with anti-His tag antibody. In attachment you can see the blot: my protein is always expressed, even in absence of IPTG (very strong basal expression). Moreover there are at least two signals, as if my protein is degraded from the start or if there were two recombinant proteins expressing in my cell colture... What do you think about it? 
-matys-
QUOTE (matys @ May 9 2008, 03:22 PM)
I've just obtained the results of the test proposed by Jipes.
I've prepared 50ml of recombinant cell colture, let it grow at 30°C and when it has reached OD 0.8 I've shifted the growth temperature at 22°C. I've collect 1ml of the culture each 30 minutes up to 3 hours, and I've resuspended each pellet directly in sample buffer. Finally I've loaded all samples in sds-page, trasferred the gel on NC and done the incubation with anti-His tag antibody. In attachment you can see the blot: my protein is always expressed, even in absence of IPTG (very strong basal expression). Moreover there are at least two signals, as if my protein is degraded from the start or if there were two recombinant proteins expressing in my cell colture... What do you think about it?
I've prepared 50ml of recombinant cell colture, let it grow at 30°C and when it has reached OD 0.8 I've shifted the growth temperature at 22°C. I've collect 1ml of the culture each 30 minutes up to 3 hours, and I've resuspended each pellet directly in sample buffer. Finally I've loaded all samples in sds-page, trasferred the gel on NC and done the incubation with anti-His tag antibody. In attachment you can see the blot: my protein is always expressed, even in absence of IPTG (very strong basal expression). Moreover there are at least two signals, as if my protein is degraded from the start or if there were two recombinant proteins expressing in my cell colture... What do you think about it?
First it's good news it doesn't look at all to be denatured and it's relatively abundant
Second. it's made without induction Ok but that's very often the case you should use special repressor plasmid for having a tough contrôl on the expression and even in thoses bacterias (like Tuner pLAC) it can still happen sometimes so not too much to worry about if your protein is not toxic to the cells
Third what looks for you like a degradation product, is really NOT for me, but rather looks like a secondary initiation site ! Check if you have a secont MET codon in your sequence after the initial one that might be used also to start the protein and explain why you would have this. It also happens to me with a malaria protein and an M.ulcerans protein
-Jipes-
QUOTE (Jipes @ May 9 2008, 02:13 PM)
QUOTE (matys @ May 9 2008, 03:22 PM)
I've just obtained the results of the test proposed by Jipes.
I've prepared 50ml of recombinant cell colture, let it grow at 30°C and when it has reached OD 0.8 I've shifted the growth temperature at 22°C. I've collect 1ml of the culture each 30 minutes up to 3 hours, and I've resuspended each pellet directly in sample buffer. Finally I've loaded all samples in sds-page, trasferred the gel on NC and done the incubation with anti-His tag antibody. In attachment you can see the blot: my protein is always expressed, even in absence of IPTG (very strong basal expression). Moreover there are at least two signals, as if my protein is degraded from the start or if there were two recombinant proteins expressing in my cell colture... What do you think about it?
I've prepared 50ml of recombinant cell colture, let it grow at 30°C and when it has reached OD 0.8 I've shifted the growth temperature at 22°C. I've collect 1ml of the culture each 30 minutes up to 3 hours, and I've resuspended each pellet directly in sample buffer. Finally I've loaded all samples in sds-page, trasferred the gel on NC and done the incubation with anti-His tag antibody. In attachment you can see the blot: my protein is always expressed, even in absence of IPTG (very strong basal expression). Moreover there are at least two signals, as if my protein is degraded from the start or if there were two recombinant proteins expressing in my cell colture... What do you think about it?
First it's good news it doesn't look at all to be denatured and it's relatively abundant
Second. it's made without induction Ok but that's very often the case you should use special repressor plasmid for having a tough contrôl on the expression and even in thoses bacterias (like Tuner pLAC) it can still happen sometimes so not too much to worry about if your protein is not toxic to the cells
Third what looks for you like a degradation product, is really NOT for me, but rather looks like a secondary initiation site ! Check if you have a secont MET codon in your sequence after the initial one that might be used also to start the protein and explain why you would have this. It also happens to me with a malaria protein and an M.ulcerans protein
I think that the second protein can't derive from a secondary initiation site, because the recombinant protein should have the his tag at the Nterm, so I couldn't see it with an anti-his tag antibody. Where does it come from???? could it derive from a premature stop in the synthesis? but there are no reasons why cells should read it once as a codon stop, once as an aminoacid...
-matys-
QUOTE (matys @ May 9 2008, 05:12 PM)
QUOTE (Jipes @ May 9 2008, 02:13 PM)
First it's good news it doesn't look at all to be denatured and it's relatively abundant
Second. it's made without induction Ok but that's very often the case you should use special repressor plasmid for having a tough contrôl on the expression and even in thoses bacterias (like Tuner pLAC) it can still happen sometimes so not too much to worry about if your protein is not toxic to the cells
Third what looks for you like a degradation product, is really NOT for me, but rather looks like a secondary initiation site ! Check if you have a secont MET codon in your sequence after the initial one that might be used also to start the protein and explain why you would have this. It also happens to me with a malaria protein and an M.ulcerans protein
I think that the second protein can't derive from a secondary initiation site, because the recombinant protein should have the his tag at the Nterm, so I couldn't see it with an anti-his tag antibody. Where does it come from???? could it derive from a premature stop in the synthesis? but there are no reasons why cells should read it once as a codon stop, once as an aminoacid...
Right I didn't remember that the His Tag was on the N termini sorry
I don't tend to believe in an alternative stop codon 
Of course if you have several clones in the mix that would be possible but with a pure plasmid that's quite unlikely.For degradation that would mean that 50% of your protein is already degraded even before induction
I've had hard time to believe it but well I've seen so many weird stuff in biochemistry and in science in general who knows in the end 
One possibility is to make a MS on the protein to see if the sequence is the good one but that's also quite some work to do
-Jipes-