Cloned genes dont express! - (Feb/14/2008 )
Hi!
I have a scFv antibody which I have made several different n-terminal modifications by using modified primers and PCR, such as in inserting cysteine, shortening the sequence before his-tag etc etc..
I have sequenced the clones and they are all inframe with the ATG (in this case pelB in pET26b) and the stop codon after the His-tag but they all fail to express. These are all identical to the Wildtype apart from the n-terminal sequence before the histag.
I have tried XL1-Blue and RosettaGami2, with 0,5mM IPTG, 37C 5hours. Ive checked both the soluble and denaturating extracts but I cant see my protein of interest at all... so if its not even expressed as inclusion bodies I guess its not expressed at all.
I have no problem with the original (positive kontroll) which expresses as usual with no problems.
Im a bit lost... could it be my plasmid vector which is the problem!? should I cut out my genes and reinsert them into fresh plasmid? can that help?
Ive read that the RosettaGami2 could just be junk and to no help at all.. true or false!?
Thanks for any kind of help or hints.
Are you very very sure that the expression construct is correct?
Is your gene any way toxic to the cell?
How are you checking to see if your protein is being expressed?
Have you tried changing the expression conditions?
Try increasing the amount of IPTG upto 1mM - 1.5mM
Try lowering the temperature the experiment is conducted at , 25 Celsius
Try adding glucose to the medium, as prevents the lac promoter from activating (due to trace lactose) prior to IPTG induction.
Use a bigger flask, for better Oxygen transfer.
If you are using LB, try using a richer medium... SOC, YT, Terrific Broth...
Do you have different isolates of your plasmid? You can try using that, on the off chance that the plasmid you are got mutated some during the construction..
Is your gene any way toxic to the cell?
How are you checking to see if your protein is being expressed?
Have you tried changing the expression conditions?
Try increasing the amount of IPTG upto 1mM - 1.5mM
Try lowering the temperature the experiment is conducted at , 25 Celsius
Try adding glucose to the medium, as prevents the lac promoter from activating (due to trace lactose) prior to IPTG induction.
Use a bigger flask, for better Oxygen transfer.
If you are using LB, try using a richer medium... SOC, YT, Terrific Broth...
Do you have different isolates of your plasmid? You can try using that, on the off chance that the plasmid you are got mutated some during the construction..
Im using LB and have tried both 37C 5h and 20C overnight and have used big shakerflasks. The gene is not toxic and I use ELISA to detect expressed protein. I know that another lab mate has tried 0,1-1mM IPTG with no change on the Wildtype scFv expression.
Im going to try to ad extra carbon source such as glucose and it might even help as u mentioned earlier, also Ive read that Sorbitol and Succrose has helped expression of scFv antibodies.
Ive never tried SOC, YT or TB but thats a good advice, thanks alot. Im gonna try them as soon as I can.
Only thing is that perhaps few parameters are not optimal but I would expect at least a little expression, if it was inclusion bodies that I would see atleast a band at appropriate size after ni-nta purification..
Ive checked the Sequence and I cant see any mutations, but maybe I should sequence the constructs again as somehting might have happened in the glycerolstocks.
Thanks for the hins, I get back as sson as I try the new media and additives.
I have a scFv antibody which I have made several different n-terminal modifications by using modified primers and PCR, such as in inserting cysteine, shortening the sequence before his-tag etc etc..
I have sequenced the clones and they are all inframe with the ATG (in this case pelB in pET26b) and the stop codon after the His-tag but they all fail to express. These are all identical to the Wildtype apart from the n-terminal sequence before the histag.
I have tried XL1-Blue and RosettaGami2, with 0,5mM IPTG, 37C 5hours. Ive checked both the soluble and denaturating extracts but I cant see my protein of interest at all... so if its not even expressed as inclusion bodies I guess its not expressed at all.
I have no problem with the original (positive kontroll) which expresses as usual with no problems.
Im a bit lost... could it be my plasmid vector which is the problem!? should I cut out my genes and reinsert them into fresh plasmid? can that help?
Ive read that the RosettaGami2 could just be junk and to no help at all.. true or false!?
Thanks for any kind of help or hints.
Tobias et. al. (Tobias, J.W., Shader, T.E., Rocap, G., Varchavsky, A., 1991. The N-end rule in bacteria. Science 254, 1374. ) reported that protein half-life was only 2 min when the amino acids, Arg, Lys, Phe,
Leu, Trp, and Tyr, were present at amino terminus, but all other amino acids conferred half-lives of more than 10 h when they were present at the amino terminus. Could this be relevant for you?
Leu, Trp, and Tyr, were present at amino terminus, but all other amino acids conferred half-lives of more than 10 h when they were present at the amino terminus. Could this be relevant for you?
Ive read about that but my protein has Met-Asp-Ile-Val at N-terminus and I dont have that problem with the Wildtype scFv.
First of all Im gonna try to put the constructs into fresh new vector from novagen then take it from there and use different media.
Leu, Trp, and Tyr, were present at amino terminus, but all other amino acids conferred half-lives of more than 10 h when they were present at the amino terminus. Could this be relevant for you?
Ive read about that but my protein has Met-Asp-Ile-Val at N-terminus and I dont have that problem with the Wildtype scFv.
First of all Im gonna try to put the constructs into fresh new vector from novagen then take it from there and use different media.
More than the last four residues could be relevant, e.g. it was reported that six leucines in the region within 20 amino acids following N-terminal Met of a protein can result in expression problems (Journal of immunological methods 2002 vol. 271 (1-2) pp. 177-84). If the N-end rule may not be relevant (and you have a his tag...) and the tips from perneseblue do not help (Glucose can make a big difference) maybe it helps to try different expression hosts. I have not used RosettaGami2 myself but maybe your mutated gene cannot be as well expressed in this host as the wildtype and this can be overcome in another host. You could try if the standard host BL21(De3)pLysS may work? You probably know this document (see attachment): have you looked for clues for which host (maybe even protein expression induction by phage) to try in the pET manual?
Hopefully your new-vector-approach will make a difference. I suggest you take samples pre induction as well as every hour post induction. You may see protein expression at 2 hours, which decreases thereafter (undetected at 5 hours): this would be a clue to toxicity. Good luck!
Well I have bought fresh vector (pET26b) from novagen and cut the new constructs into the new vector and still I get no expression at all compared to the wildtype.
Here is the only difference in the C-terminal compared to the wildtype, not that the N-terminal is identical to the wildtype.
Wild Type pelB-MyGene-RRRPHRIPAAALEHHHHHHstop
New One pelB-MyGene-GLEHHHHHHstop
New One pelB-MyGene-GGCLEHHHHHHstop
and so on a 10 more constructs with different C-terminal, longer and shorter ones. NONE expresses.
So basically the only thing I have done is to remove this part RRRPHRIPAAA, which comes from the pET26b vector.
The wildtype expresses and binds to its antigen, in both LB and TB media, induced at 37, 30 and 20degrees but the new one doesnt. I dont even see anything in the denaturating prepps.. so no inclusion antibodies. Ive used Rosetta, RosettaGami2, BL21 and BL21plysS for expression.
Ive sequenced the new one 4 different times and there is absolutely no mutations and they are inframe with the pelB and the His tag.
Now I wonder how the wildtype is expressed but the 10 other ones dont at all!?!? 
I wonder if its a vector, plasmid issue!? Host issue?
I use XL1-Blue as cloning host, I use NEB restrictions enzymes and I cut my vector and purify it from agarose gels before ligating with minimum exposure to UV light etc.. ( I use the NucleoTrap http://www.mn-net.com/bio/Products/Nucleic...S/Default.aspx)
I also wonder if something is happened with the wildtype vector (long time ago) which makes my protein to express from the beginning so maybe I can use the wildtype prepp and cut out the gene and insert the new ones in the wildtype vector and see what happens!?
Im lost!