Expression problem in pQE30 vector system - (Nov/25/2005 )
Hello !!!
I have been working with 1.56kbp gene and attempted to express my gene in M15 competent cells ( pQE30 is the expression vector).6xHis is located in the `N` terminal side .I could not get any distinct HisTag protein after expression using HisTrap column.My expected protein size is 55.7Kd , where as i was getting lot many non specific bands even after HisTrap column purification and one band consistantly at 66Kd range. With the above doubt , i checked the insert sequence and its alignment.I also confirmed the vector and insert size using control. In PCR confirmation,Insert specific primers could amplify my gene of interest as well to the expected size.
To confirm whether my Gene of interest could express protein or not even in very low concentration, i performed Western blotting using AntiHis Monoclonal Antibody . A known His Protein was used as control sample .Unfortunately I do not find any His protein expressed in this case however control shows clear band.
I am wondering , what could be wrong?If every thing is correct , why the Gene is not getting expressed with pQE30 Vector????Is there anything to do with the media where I grow M15 cells? Please find a Gel pic with various purification stages and last 4 bands are Histrap column purified active fractions. Any suggestions will be appreciated.Thanks in advance.Sincerely
Saikat Chakraborty
Mie University , Japan
I have been working with 1.56kbp gene and attempted to express my gene in M15 competent cells ( pQE30 is the expression vector).6xHis is located in the `N` terminal side .I could not get any distinct HisTag protein after expression using HisTrap column.My expected protein size is 55.7Kd , where as i was getting lot many non specific bands even after HisTrap column purification and one band consistantly at 66Kd range. With the above doubt , i checked the insert sequence and its alignment.I also confirmed the vector and insert size using control. In PCR confirmation,Insert specific primers could amplify my gene of interest as well to the expected size.
To confirm whether my Gene of interest could express protein or not even in very low concentration, i performed Western blotting using AntiHis Monoclonal Antibody . A known His Protein was used as control sample .Unfortunately I do not find any His protein expressed in this case however control shows clear band.
I am wondering , what could be wrong?If every thing is correct , why the Gene is not getting expressed with pQE30 Vector????Is there anything to do with the media where I grow M15 cells? Please find a Gel pic with various purification stages and last 4 bands are Histrap column purified active fractions. Any suggestions will be appreciated.Thanks in advance.Sincerely
Saikat Chakraborty
Mie University , Japan
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Well welcome to the WWTERP (Wondeful World of Trouble in Expresion of Recombinants Proteins)
More seriously not all proteins will be right away expressed in any kind of system
Any protein is a given problem in it's own the rule doesn't apply for all proteins
I am currently working on Myccobacterial proteins and was able to generate two verynice functionnal proteins but the new ones I'm currently on are not expressed at all even if I use the same vector same type of recipients expression host methods etc....
What can you do ? Good question
Try other expression host
shorten your protein if possible
Check carefully the adjacent codon following the original Met because of the instability
Novagen is giving this explanations
[color=#FF0000]Arg, Lys, Phe, Leu, Trp or Tyr gave a half life of only 2 minutes !!!!!!
The worst codon is Leu
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Check your codon usage some rare amino acids are only generate in very small amounts by E.Coli leading to very poor expression (Arg, Leu, Gly and Pro )
Some proteins need a disulfide bonds so use an expression host like Origami or Rosetta Garni
Lower the temperature ofthe culture might help to reduce problems
use less IPTG
Add 0.5 to 1% Glucose to the culture
etc.... the list is too long I recommend all persons having troubles with expression to go to the Novagen website and download the pET Blue system Manual TB249
Novagen website
hope it helps
pesji
HELLO!I have a question.I had inserted my aimed gene fragment of 480bp into the vector of pQE30.But I found the sequence before the site of SphI which was forward enyme was not coincident with pQE30 .Is it worry in the vector of pQE30 ?