I am currently cloning selected antigens from Acinetobacter on Qiagen's pQE31 vector and ecoli M15 as host. Cloning is successful but "no" expression visualized after SDS run taking in consideration bands did show in expected size after western blotting .I changed induction conditions(inducer conc./temp./time) with no results using IPTG as inducer.
i also tried shifting to a different expression host JM109 with no results either.
Hi Abdulrahman,
I don't know the protein's molecular weight but if it is smaller than 10 kDa you can consider instability. (The QIA expressionist, 06/2003, p52)
The another possibility could be that you chose a colony which expresses a low amount of the protein. If you use PCR for colony selection, I suggest you use dot blot to determine the high expression colonies.
Good luck.
Hi Cigdem,
Thank you for your answer.
I am actually working on several antigens ranging from 16 kDa reaching 40 kDa
I did apply dot blot technique to determine optimum induction condition using Anti-His tags, it did show positive His tag at different conditions yet after scaling up growth (1 L) at these selected induction conditions it also refuses to show on SDS gel as well. I could not find an explanation to that.
I followed QIA expressionist troubleshooting without any sensible explanation other than that proteins may be toxic.
Do u think shifting the pQE vector to others as pET for instance would do the job ??
really grateful for ur time and concern thanx in advance
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