I am having trouble in expressing Streptomyces protein in E.coli. I had similar problem in past with another protein. I started with two genes and I could get protein for one but the other one was not successful. now I am trying again for another protein. I started his 8 tag purification with around 30 mg total protein. At the end I could not get any purified fraction of protein. I am using pET28b plasmid and 0.5 mM IPTG for induction.
Was the protein expressed? You can check by SDS-PAGE and, if necessary by Western blot using anti-tag antibody.
If it was expressed, was it in the soluble fraction or the pellet when you centrifuged the cell extract?
yes the protein was expressed. mainly it was in pellet. here I have attached the SDS-PAGE gel. S1 is before induction and S2 and S3 are after induction. Similarly P1 is before induction pellet and P2 and P3 are after induction. Loading the pellet was difficult and it made gel little messy. Pass is pass through, W1 is 1st wash and W2 is 2nd wash. E1 and E2 represent 1st and 2nd elution. The protein size is 35.91 KD.
I am not really sure which band corresponds to your protein of interest. First you should check by sequencing if the His tag is really in frame with your protein. The washing fraction gives little information on SDS PAGE. More interesting are the Nickel-NTA beads before and after Elution. So take maybe 10µL of the beads, boil it in SDS-PAGE loading buffer and analyze the beads in SDS-PAGE. If nearly all your protein is in inclusion bodies try to reduce the temperature during expression to room temperature or 18 °C. If the protein is still in inclusion bodies the only way is to resolve the insoluble fraction (in 6M GuHCl or 8M Urea) and refold the protein.
It seems that your induction didn't work,basically from you gel,so maybe you could add a positive control in your next experiment,and if control expressed well,then the problem is your clone,recheck it carefully.
Most of streptomycete genes are hard to express in E. coli due to the high G/C content and rare codon usage. Sometimes adding a large tag such as GST or maltose binding protein helps. You can also try to co-express with chaperon proteins. Another way is to codon-optimize your gene, but I have found most of time this method doesn't really help. If everything fails in E. coli, you can express your gene in streptomycetes. There are several expression vectors available. This will give you soluble proteins, but you have to give away the high yield. Good luck.
Thanks everyone. I have already checked the sequence and there is no mutation or frame shift. @Rongfeng Li Thanks for your kind suggestions. yes I agree, its relatively difficult to clone Streptomyces genes in E.coli. I started with 4 genes. One of them was luckily expressed. I have done expression in Streptomyces and if finally I could nor ge this protein from E.coli then I have to move towards that option.
Hi, Courtney.
Regarding the 72h growth, you don't add IPTG, right? Just let the culture grow for 72h and collect the cells? And what temperature you usually grow the culture?Thanks.
Hi Ammara
Did you use proteases inhibitor during sample preparation for affinity chromatography?
some times the unstable protein get chop off by the proteases of bacterial cells.
you can also do the peptide mass finger printing of your desire band in order to check that either your protein has expressed or not.
Thanks
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