problem in protein expression

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dalim2000
dalim2000's picture
problem in protein expression

Hello
I am having problem with expression of gene. I got 2 clone, my first clone is 369 bp which is well expressed. When i made another clone by adding 63 bp of positive charged amino acid with my first clone, i cant expressed it. I am using DE3 cell for expression and my vector is pRSETA which has his tag protein.
I tried in different condition like temperature and time of induction by IPTG. Could you give me any suggestion for this.

regards
Dalim

Chin Fen Teo
Chin Fen Teo's picture
Hi dalim2000,

Hi dalim2000,

When you mentioned you couldn't express the clone with the extra +ive charge AA, what kind of samples you were looking at? Total lysate or S/N? And what kind of conditions you have tried so far??

dalim2000
dalim2000's picture
Hi Pippuri

Hi Pippuri
Thank you for your reply with quary. I did seeding of transformed cell into LB broth and incubated at 37 degree until get OD 0.4-0.6, then added IPTG 2mM/ml and incubated further for 12 hours at 30 degree in shaking incubator.
I checked the total lysate by SDS page. I did not find my expected expression. I also tried different temperature like 25 degree and 37 degree for different time, such as 5 hours incubation after adding IPTG.
Thank you again. Looking forward to hearing from u.

Dalim