protein expression in bacteria
Joao Goncalves
Friday, 14 March 2008 18:17 UTC
Hello,
I need to produce a protein in bacteria and have no experience in that field. I need the protein to produce an antibody against it.
I’ve cloned its coding sequence in the mammalian vector pcDNA3. This vector has the T7 promotor…does any one know if this is enough to express the protein in bacteria…or should I clone it in a proper bacterial vector?
Thanks in advance.
João
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Replies
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I have no idea what you are talking about..but it sounds very interesting.
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Hi Joao
I dont know why you want to use a mammalian vector for this purpose. However, from my own work I use pET21b from Novagen it is quite good expression system or you can use pQE expression system from Qiagen . Both are for bacterial expression of recombinant proteins.
Hope this helps
Regards
Farasha -
Hello João,
I don’t know if you found the answer to your question already, but I would like to suggest you a few things.
Use pET bacterial expression vectors. You can find a variety. The basic differences are on the location of purification tags and Multicloning regions. These vectors have a selective marker (antibiotic) and also they provide you a strong promoter upstream of your gene. This promoter is able to be regulated by IPTG. So, you will grow your cells until OD600 0,6-0,8 and then you need to add 0,4-0,8 mM IPTG in order to induce the expression of your protein.
You don’t want to have expression from the begining because it might interfere with the growing of your bacteria. In addition, I would suggest to use bacteria strains which are desinged for protein expression like Rosetta2 or BL21 pLysS.I hope this will help,
Maria
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Thanks a lot for your replies.
I have never done this and was wondering if I could use the same construct I already have for mammalian cells since it has a T7 promoter…now I know I can’t because this vector doesn’t have a Shine-Dalgarno sequence… the T7 promoter is for in vitro transcription.
Now I’m trying to clone my gene in a bacteria vector pGEX…but I’m not being very lucky….but I hope I can get the clones very soon to produce the protein and get an antibody.
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First, don’t use a mammalian expression vector to produce a protein in E. coli. As Maria Noutsou pointed out, you are better off using a system specifically designed for production of proteins in bacterial system.
The pET system, which employs the phage T7 promoter, is among the most popular. There are many different versions of plasmids that you can choose from depending on your needs. However, this promoter is so strong that more often than not the recombinant protein will accumulate as inclusion bodies. If you can purify the protein under denaturing conditions, this can be great. However, if you need active soluble protein, then you might need to refold the protein which can get tricky. Several vendors (Invitrogen, Novagen, Takara, New England Biolabs, Qiagen, Promega, and yes us too, AthenaES, sorry for the commercial) have alternative expression strategies to the pET system that also come with different affinity tags to simplify purification. We have successfully used the pET, pQE, pMal, pGEX, pTrc, pKK as well as our own systems. I suggest that you take some time to learn about the different varitions available and how they would work for you particular needs.
After you select an expression system and start producing the protein, be wary of the expression conditions you employ. The stain, medium composition and induction conditions (inducer levels, induction duration, culture temperature) employed can all affect the accumulation levels.
Good luck.
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Thank you all for your replies. I’ve already cloned my gene in a pGEX vector and already tested if the construct was working and it is. The protein seems to be both in the soluble and insoluble (probably in inclusion bodies) fractions. Hopefully I’ll be able to purify my protein and produce an antibody against it.
Best wishes.
João
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