I am overexpressing a protein BL21 ecoli cell. The total protein content after cell lysis is about 50% of the wet weight (crude cell pellet). Isn't this bit high??
The total protein content about 50% is OK. I would expect even more...
Bacteria are packed with proteins and nucleic acids, at much higher ratios to fresh weight compared to animal cells and much much much higher than plant cells (which are mostly filled with an aqueous vacuole). Perhaps 50% of fresh weight is a bit high, and I'm not sure how you have determined it and if you have first washed the liquid broth away before you lysed the cells...
I wouldn't be too concerned about total protein content. What matters is if your particular protein that you wish to overexpress is sufficiently abundant compared to endogenous proteins so it stands out on a coomassie stained SDS-PAGE. The other thing that really matters is whether your protein is soluble or stays in the pellet. The answers to both these questions are found when you lyse BL21 cells with empty vector (negative control) and your overexpression plasmid (your test sample), spin cell debris down to get supernatant, then re-solubilise the pellet by sonication with an equal volume of sample buffer, and then boil the 4 samples with an equal volume of SDS-PAGE sample buffer mix, and run on SDS-PAGE for Coomassie staining. This volumetric way of working allows you to see if the protein is expressed and if it is soluble. You want an extra band in the test sample in your first supernatant. If you don't see anything new there, you may see an extra band in the solubilised pellet (means the protein is insoluble, i.e. found in inclusion bodies). If you don't see an extra band anywhere, then your protein is not sufficiently expressed or not expressed at all..
There is no "correct level of over-expression". It is entirely dependant on the individual protein - some express poorly, others well - some can be cytotoxic and have detrimental effects on the cells, others are totally harmless. A lot of overexpressed proteins end up in inclusion bodies which may be difficult to solubilise. Having a high level of expression does not necessarily convey an advantage if the product is largely insoluble except in 5M Guanidinium or 8M urea. The most important criteria is ultimately, functionality, rather than the total amt. A large amt of incorrectly folded inactive or insoluble protein may not be of much use except to raise antibodies.
Thanks for your valuable suggestions. My protein is insoluble in nature, i.e. found in inclusion bodies. Huge amount of overexpressed protein is seen in the pellet fraction after visualizing in SDS-PAGE.
After induction i have harvested the cells by centrifugation and decanted the media and taken its weight (wet weight). Then i have lysed the cells by both chemical and mechanical methods and quantified the total proteins before separating into supernatant and pellet fractions
However my query is regarding the total protein content in a typical overexpressed E coli cell. If the wet weight crude cell pellet is 100mg and after its lysis (sonication) its total protein conentration is 50mg/Lt.culture...is it fine??
It is possible to get very high expression. As I don't know the volumes of your sonication I have no idea of the yield or what the protein conc and amts are in the final supernatant and pellet fractions ( you have stated an amt wet weight of 100 mg, and a concentration of total sonicate of 50 mg/ml of culture).
Thank you Mr.Ian. There are few corrections here!!!
From 25 ml culture, i got 100mg crude cell pellet (wet weight)
I dissolved the cell pellet in 1.5 ml of lysis buffer and sonicated and quantified (33.33 mg/ml). Thus the total protein i calculated was 50 mg.
Kindly help
From what you have written, am I correct in : 1) the crude wet weight of cells from 25 mls was 100mg. This includes protein DNA RNA etc. and is not an accurate measure (dry weight) 2) The 100 mg was sonicated in 1.5 mls lysis buffer and the total soluble? protein concn determined as 33.3 mg/ml giving a protein amt of 50mg (the rest being DNA, RNA, insoluble material?). This is not a measure of the cloned over-expressed protein, only the total soluble cellular protein. To get the % over-expressed protein you would have lyse cells, purify the inclusion bodies (from the "insoluble materia), determine the protein concn and amt., in the soluble and insoluble fractions and from that detemine the % expressed protein assuming it was all in inclusion bodies.
Thank you very much Mr. Ian
You are correct that we cannot measure the amount of overexpressed protein from just calculating its cell pellet weight and cell lysate.
However i had the query on whether the amount of protein (50mg) from 100mg wet weight cell pellet is logical?? From my study it shows that 50mg of protein from 100mg of cells (though roughly)..Can a bacterial cell accomodate about 50% protein in it (even though overexpressed)
If you want to make antibodies, then the presence of your protein in the pellet can be used as a simple method to remove 80% of other proteins because they are soluble. When you then boil the pellet and load on a gel, the protein of interest is a major band. In the past we have made excellent antibodies by simply loading an entire gel with the comb in reverse (one massive slot), making sure it is 100% horizontal and avoiding overload, and then running the gel slowly and cutting out the major band after coomassie staining. We ground up the blue SDS-PAGE strip with liquid nitrogen, pestle and mortar, and sent the powder to Eurogentec for immunizing rabbits. Antibodies were excellent, probably because the protein was denatured and the polyacrylamide helps to spice the immune response up (like Freund's adjuvant). .
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