hello everyone
I am trying to express a protein with SUMO tag which used to express initially in buckets by a labmate in BL21 RIL at 500mM iptg induction overnight at 18*C.
but when i tried expressing the protein, i am getting more of SUMO and very little of my protein.
I have verified the plasmid with RD and tried expressing with fresh plasmid. Also i screened with other expression cells. at 20*C as well as 18*C. All i get is sumo tag in bulk.
I know that the protein expresses but why its not working am not able to decode.
Please help me with some ideas to increase the expression.
I would not trust the restriction digest analysis alone, it would be best to send the plasmid for sequencing to be absolutely sure that your insert is still present. BL21 RIL is not a recA- strain so you might actually lose your insert (in case the freshly isolated plasmid comes from the same source).
I presume that the use of SUMO and low expression temperatures serve to obtain the soluble protein; however, if the protein is expressed at 30 or 37 C this can at least allow you to visualize that it is being synthesized in full length (running SDS-PAGE with cell extracts after a shorter period of time). Also, overnight expression might mean different amounts of time for different people; so it might be the difference between 12 or 16 hours. Ideally, you would track the synthesis of your protein by Western blot over time to make sure it is not getting degraded at some point... In addition, 500 mM IPTG seems a little bit excessive, usually 1 mM IPTG is the highest concentration used.
Dear Ana
Thank you for your valuable suggestions. I have straightaway sent it for sequencing.
We use 0.5mM IPTG final conc. My mistake i mentioned the stock conc.
Hi, I am facing same problems as mentioned in above raised question. I am using BL21DE3 strain for expression of my two genes by pet-sumo vector. But I am getting only Sumo bands while my SDS-PAGE.
We use baffled 125ml vessels with only 10ml culture in them to provide proper aeration. And we culture the cells at 200RPM in shaker incubator. I start from by culturing the fresh plate always. I use 0.1M IPTG in final volume as per written in your given protocol. We prefer to give induction between 0.4 to 0.6 OD.We have confirmed the presence of intact plasmid by plasmid PCR with gene specific primers and orientation of inserted gene by using T7 reverse primer and gene specific forward primer before proceeding to the 1st batch of expression and then we repeated our expression almost 6 times. I usually load 20 to 25 microliter sample in each well of SDS. Help regarding above problem will be highly appreciated.
You can try running a Western with anti-his antibodies to determine optimal conditions of expression for your protein (I would normally try 37, 30, 25 and 18 C and collect every hour or a couple of hours for low temp).
However, pET-SUMO constructs have the N-terminal tag, so if you cannot get rid of the truncated product you will need to move the tag to the C-terminus in order to separate the two. Alternatively, you can use a second tag such as FLAG/Strep; two rounds of affinity purification would then give you a very clean product. If you want you protein tagless, just include a protease site preceding the C-terminal tag. Good luck!
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