I have cloned lipase gene into pET28+ and express it in Rosetta(DE3)pLyss. I have tried to induce with IPTG but I can't spot any difference between the clone and the empty pET. I varied the temperature, IPTG concentration. I have tried whatever I could possibly think of. I happened to read Studier (2005) on the autoinducting medium. I carried it out at 20C O/N. The expected protein size should be around 23kDa but I am seeing a band at around 35kDa. Could someone enlighten me please?
Thank you everyone for your feedback!
The construct has already been sequenced and the reading frame has been checked. I am using BamHI and XhoI. I am actually trying to express a gene from Rice in E. coli, so I am worried that it might be toxic. But I am not sure if it is really toxic to the cell. I thought it is always good to use pLysS. I will try to go for purification next.
Hi Shanice!
I guess your plasmid will add a His-Tag. Have you thought about confirming the expression with an anti-His antibody ? (e.g. anti-penta His, which is one I used before) Then you know where your protein is in the gel and this detection method will facilitate optimization of expression (e.g. through extended expression times etc.).
Best regards!
Christian
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