I want to raise the antibody against a membrane protein which is of the size 180 kDa. I already have a clone in pET vector. Is there any chance to get a reasonable amount of expression, given the size of the protein. Is there any protocols for the expression of proteins of large molecular size.
Thank you
Dear Jibin,
the size of your protein can be not necessarily a problem. What instead can become an important issue is the structure of your protein. Are any prostetic groups present or any idea that metals could be part of the protein structure? Another problem can arise from cysteines. If a large number of cysteine residues, and consequently of disulfide bridges, are present then your protein is likely to encounter folding problems and end up, due to disulfide bridges random shuffling, being a very unstable and unsoluble product. This may happen even if only a restricted portion (a domain or subdomain) of your protein would have such features.
In general, if a great number of posttranslational modifications are present then the more complicated will become to express a protein in E. coli. For the expression of a protein with multiple disulfide bridges (8) once I tried the Origami E. Coli strain without achieving a great success I must say. However, you may encounter particular folding problems even in the absence of many cysteines. In this case, there is no a magic suggestion to express any possible protein. It may help to play with the concentrations of IPTG or also try different temperatures of expression (usually lower temperatures, 28-30 °C, may favour the folding process). Another suggestion I could give you is to use BL21(DE3) codon plus strain that is highly recommendable especially if you have cloned a cDNA in your pET vector. This should solve any troubles coming from the E. coli codon usage.
However, of course your main problem is that you are dealing with a membrane protein (that will be therefore highly unsoluble and probably quite difficult to purify even if overexpressed). Why don’t try and express only a portion of your protein, cytosolic or extracellular, that you can then exploit for raising antibodies? From the computational analysis of your protein structure, and of possible domains present, you may get useful information to narrow down a portion that could be used (you may find interesting and freely available software at the www.expasy.org site). In this case, it is safe to imagine that you will end up working with a protein with a smaller size than 180 KDa.
What else I could say? Good luck!...In order to give you some more advice it could be also helpful to know which protein you are after…:)
Dear Jibin,
the size of your protein can be not necessarily a problem. What instead can become an important issue is the structure of your protein. Are any prostetic groups present or any idea that metals could be part of the protein structure? Another problem can arise from cysteines. If a large number of cysteine residues, and consequently of disulfide bridges, are present then your protein is likely to encounter folding problems and end up, due to disulfide bridges random shuffling, being a very unstable and unsoluble product. This may happen even if only a restricted portion (a domain or subdomain) of your protein would have such features.
In general, if a great number of posttranslational modifications are present then the more complicated will become to express a protein in E. coli. For the expression of a protein with multiple disulfide bridges (8) once I tried the Origami E. Coli strain without achieving a great success I must say. However, you may encounter particular folding problems even in the absence of many cysteines. In this case, there is no a magic suggestion to express any possible protein. It may help to play with the concentrations of IPTG or also try different temperatures of expression (usually lower temperatures, 28-30 °C, may favour the folding process). Another suggestion I could give you is to use BL21(DE3) codon plus strain that is highly recommendable especially if you have cloned a cDNA in your pET vector. This should solve any troubles coming from the E. coli codon usage.
However, of course your main problem is that you are dealing with a membrane protein (that will be therefore highly unsoluble and probably quite difficult to purify even if overexpressed). Why don’t try and express only a portion of your protein, cytosolic or extracellular, that you can then exploit for raising antibodies? From the computational analysis of your protein structure, and of possible domains present, you may get useful information to narrow down a portion that could be used (you may find interesting and freely available software at the www.expasy.org site). In this case, it is safe to imagine that you will end up working with a protein with a smaller size than 180 KDa.
What else I could say? Good luck!...In order to give you some more advice it could be also helpful to know which protein you are after…:)
We have used DE3 along with specific added tRNAs for modifications before (available commercially from Stratagene), a good alternative is the Rosetta series of strains. Both can express proteins of higher molecular weight. But i would caution you against expressing membrane proteins in soluble form as most of it would go into inclusion bodies. If your protein is eukaryotic in origin, then i would recommend you use a eukaryotic expression system - picia or baculovirus.
As pointed out by other contributors, you may end up expressing insoluble protein, which you may likely use the protocol for inclusion body isolation and solubilisation. This can then be dialysed in descending conc. of urea. If you are interested in using the protein in order assays aside from antibody production, I would suggest you express part and not all of the protein bearing in mind the portion that is of interest. But generally, I have succeeded in expressing membrane proteins that are
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