Hi,
I have cloned a protein in pET 28a and expressed in Rosetta 2 cells. I could detect the protein with anti His antibody and also protein specific antibody in western. However on purification, I could not get yield. I have also checked with whole cell protein where I could not find large difference between the uninduced and induced cultures. Is there any means to increase the expression or yield.
I have tried with various expression conditions like lower temperature for over night, lower concentration of IPTG, induction at 37 deg for 3 h with 1 mM and 0.3 mM IPTG. I could not find much variation in the expression.
Could you suggest what can be done further.
FYI: the protein is active after Ni elution (0.8 mg/mL protein from 1L) and even after gel filtration (0.6 mg/mL where I have loaded 5 mL of the Ni fractions).
Thank you.
You can actually optimize the expression condition by varying the post induction temperature, post induction time and amount of inducer.
you can try with other bacterial strains for protein expression such as C41, C43 or BL21. or clone the gene into other expression vectors.
hi
try inducing cells at OD600=0.4 and 20 °C overnight using 1mM IPTG. if does not work properly, you should think about changing your plasmid vector... good luck!
Are you using Rosetta 2 (DE3) or only Rosetta 2 cells? Please take note that there is a vast difference between these two competent cells. The latter does NOT carry chromosomal copy of the T7 RNA polymerase. As such, pET vectors cannot be used as most of the pET vectors contain T7 promoters within their vector backbone.
Try checking your expression host first. You can find further information about the host cells here:
http://www.emdmillipore.com/INTL/en/product/Rosetta™-2-Competent-Cell-Set,EMD_BIO-71405
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