Problem in protein expression
Posted 09 January 2005 - 06:09 AM
I am trying to express a mammalian protein of about 20 kD in BL 21 . I tried different temperature( 30 & 37) and different IPTG concentration ( 1mM, 2mM & 3mM). The protein is not expressing at all.
The protein had been cloned iin the my lab and got good expression. But I couldnt get the expression. The vector is pQE30. I am new to these techniques. Please give your suggestions
Posted 11 January 2005 - 03:49 PM
First are you sure that the cell line used for the protein expression was BL21(DE3) It needs to be a DE3 cell line for expression to occur?
Given that it is a mammalian protein I am suprised that the BL21(DE3) cell line was used, I would have choosen Rosetta(DE3). What exactly are the conditions that were used by the previous person that did the work on it.
Did you look to see if the protein was in the insoluble portion of your cell expression? Or was is really not expression at all? (neither in the soluble or the insoluble fraction)
Hope this helps to get you started.
Posted 13 January 2005 - 06:12 AM
Thanks for your suggestions.
The BL 21 cells I used was used by others for mammalian protein expression and worked quite well. The protein which I am trying to express also previously expressed in BL 26 . The temperature is 37 and the IPTG concentration is 1mM. I tried to express the protein in BL 26 as well as BL 21 but didnt work. I sequenced it and the sequence is fine and also in the frame. But couldnt get the expression even at the basal level.
So donot know how to proceed......
Posted 13 January 2005 - 09:02 AM
I would try M15[pREP4] from Qiagen, because it original strain for this vector. Or co-transform plasmid pREP4 into strain of your preference. You need this plasmid, when you express protein in pQE30-family, because it contains repressor, otherwise you never get regulated expression – it will express constantly. M15[pREP4] already has this plasmid. And by the way, you don’t need (DE3) strain for pQE-family, since expression in this vectors driving under control of T5 promoter, which use host RNA-polymerase.
So, I think you have expression at middle level, but since you have not pREP4 in your cell it is constant, and you can not see on the SDS-PAAG. Even if expression very low now, I believe using of repressor plasmid pREP4, can solve your problem. For more information, please, check www.qiagen.com and download “Qiaexpressionist” manual. Hopefully, it can solve your problem.
Posted 16 January 2005 - 07:15 AM
I will try to get pREP4 for further experiments. Still I have some doubts. I did westernblotting in the cell extract ( induced with IPTG and non induced ) and couldn't get any band in it. I used a positive control(protein) also and it formed fine band. Still there is a chance of low expression??
Posted 18 January 2005 - 08:16 AM
If this protein was expressing before in the lab in the hands of someone else, that person should contribute to finding a solution for you- are those cell lines and constructs available to you? If the person is not available, have the supervisor help you. Young researchers suffer when their seniors are less than detailed about their methods.