I am trying to over express two peptides 8 K Da and 7 K Da, in E coli 2DE3 Rosetta, using pET28a+ vector. I used LB media, induced the cell at 0.5mM and 1mM IPTG, using the OD 0.8. I induced the cells at 18C, 25C, & 30C. But unable to express my proteins. After 4 hours of induction I lysed the cells using 20mM Tris-HCl, 3X laemalli with beta mercaptoethanol, 95C for 10 min, but unsuccessful. I used another lysis buffer 20mM Tris HCL, 1 mM EDTA, 0.1% Triton-X 100, Lysozyme 1mg/ml, I treated cells for 15 min and then fastprap for 2X speed 6.5/30 sec. Centrifuged 12000 rpm/5 min, treated with laemmali and beta mercaptoethanol then 95C for 8 min and on ice and loading to SDS PAGE. But still I have not found any difference in band between empty vector and inserts. Why my peptide is not over expressed? or do I have used correct method to break open the cell? The peptide is 40% hydrophobic.
do you have an antibody for western blot? and you can use tricine gel if u need to resolve it better. if nothing works I would go for fusion tags.
Yes, my both peptides have his-6 tag C-termini. No signal peptide. I also did westernblott with anti-his Abs. but still no band for overexpression. I checked the sequence of ORF and it is fine. I always used 18% tricine gel.
try different host cells and different induction times and temperatures....if these don't work use a different vector with bigger fusion tags such as GST MBP etc
Hi Nimerta,
Did you use chloramphenicol in the media? What's the codon composition of your protein? Usually Rosetta strains are used for proteins with eukaryotic codon composition, so if your protein has a bacterial codon composition you can try use another strain. Also, have you checked if your peptides are in the insoluble fraction? Induction at 18 deg C should help avoiding inclusion bodies, but that is not always the case. I would still check the insoluble fraction, and if they are there you could try to change promoter if yours is very strong.
You are working at the dye-front bundary of the gel. Yes, 18% gel will help a lot, but if you are not careful you can run you protein out.
1. Why dont you just run the crude extract on Ni/Co-HisTag affinity gel - even 2ml column is fine. Wash unbound with 10 mM Imidazole, then just batch elute your bound proteins with 300 mM imidazole. Now you have removed a lot of "background" protein and you can see you protein more exclusively. DONT FORGET TO PUT FULL PROTEASE INHIBITORS WITHOUT EDTA.
You can even get small spin columns for Ni/Co-HisTag agarose and do several samples at once. This is very fast.
http://www.zymoresearch.com/protein/his-tagged-protein-purification/spin-protein-miniprep
http://www.clontech.com/US/Products/Protein_Expression_and_Purification/His-Tagged_Protein_Purification/Cobalt_Columns-Spin
Given that you hopefully used MW markers, and your proteins have not run off the gel, there are still the two issues I think that are important to address before changing the codon usage, strain, etc. The first is that we typically used an o.d. of 0.4-0.6 and then 4 hr at 37 degrees or overnight for 16 degrees. Second, your protein may be in IBs, as Aleardo suggested, so adding 6M urea to the lysis buffer may be advantageous to ensure you aren't missing anything because of your lysing conditions. I liked the spin columns too as Prem has suggested.
1. You can make your own by buying the empty columns that are cheap and add about 0.3ml of Ni NTA agarose.to the 1 ml columns, washing with 1ml, and then eluting with 0.5ml, 500mM imidazlole.
2. You can enrich for your protein and purify other stuff away in one step.
3. You can do things in a high throughput fashion and try multiple expression conditions.
Thank you all of your nice suggestions. I also tried for Ni NTA prification under denaturing condition ( 8M urea) , and my ladder band of 1.4 KDa was there so I am sure my peptide is not run out. But still no difference in band between empty vector and my insert vector lysate.
Did you try Ni affinity under normal conditions...no urea etc.?
Its possible then that expression is very low?
Do you choose multiple single colonies and grow them individually...pooling colonies is a mistake? Grow 5ml of each and analyze...
Are your bacteria freshly transformed? In case of problematic proteins it is the good practice, takes not much time.
Maybe you are losing the plasmid. I agree to freshly transform and I don't know much about strains, but rec A minus is best. Sorry, to not be of much help.
I think you should try to fuse your peptide with MBP, it will increase solubility and expression.
thank you all of nice suggesstion but I have tried all these listed conditions as well as GST tag using pGEX4T-3 vector and Ecoli BL21 DE3 for expression. The sequence is fine.
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