I am trying to express mutants of my protein whose size is 30 Kda... however following purification by Ni affinity column ... the SDS PAGE shows the mutants appearnig at size 20 Kda.. is there a reason for that.. knowing that the colong PCR shows bands at around 800 bp which is equivalent to my protein.. how can the mutant has the same size in PCR and shows different size in SDS PAGE?
Dear Sherif.
Was the non mutated protein able to run at the correct mw? about 30KDa?
because in this case, if your sequence us ok and you do not introduced any new STOP codon during your mutagenesis a so strong reduction may be associated to protein degradation that may happen because the mutation destabilize your protein conformation and make your protein more susceptible to proteases.
Do you have a n- or c- term his tag?
probably the best way to check it, is send your sample to mass spectometry analisys by determinino or intact mass , or peptide fingerprint after trypain digestion or n term sequencing of your già tag is located at the c- term. All those approaches can provide you usefull information but may differ on cost and sample preparation.
Check if in your university there is a mass spectometry facility or group able to handle proteins and discuss the best approach with them on the basis of their expertise.
good luck
Manuele
for the non mutated protein yes it appears at the correct size
I don't know if this make a difference or not but the non mutated one is inserted into PQE
while the mutants are inserted into pet28a
Hey Sherif
If the mutant is 30KDa what is the size of of the Wild Type and also check if your protein has some post translation modification and if its prone to proteolysis.
Dear Sherif.
did you checked the sequence of your mutant or you only performed the PCR screening? In case not, check the sequence.
Do you use the same E.coli for expression or different? Because while the vector probably can affect more the expressio level than degradation, different E.coli strain can also have a role in different degradation.
Where is your mutation located? At-Nterm, C-term?
MAnuele
dear Manuele Martinelli
i checked the sequence ..
i find out that beside the desired mutations ..there are other changes in the sequence.. where does this come from ? What could be wrong ?
I received the primers ..did pcr site directed mutagensis..cut using dpnI ..trasnformed into dh5alpha .. purify again and then send to sequencing .. where is the problem ?
Are those changes close to the primers annealing regions?
did you used an high fidelity polimerase for amplification of your insert by pcr?
No they are far
i use primestar max
my colleagues use same method for mutation of other enzymes and they were successful so the problem is with me and i dont know why
Dear Sherif
it is not simple to give you hints.
Problems localized close to mutation site and therefore close to primer annealing region can due to mistake on primer design or to problem on primer annealing and sometimes in the latter cases is enough to sequence more colonies and is it possible that you find a variability in colony sequence (some good, some wrong)
if the change are far from the primer annealing site, it seems or an error if the polimerase or a problem already present in the template.
i need at least to know more detail about all the protocol follower from wild type to mutant. ifi understood well this are on 2 different vector pet28 and par so you did not performed a direct mutagenesis approach by performing a single pcr of the vector on the ed template but you did also an intermediate sublconing step. i'm right? Was the sequence of the intermediate correct?
however for the Next future i suggest ti you to evaluate PIPE cloning ad approach for mutagenesys.
check a presentation on my blog: ProteoCool https://proteocool.blogspot.com/ at PAGE 1 cloning
it is quite simple and succesfull in the most cases of you optimize the dna template to eliminate the background.
Manuele
Dear Sherif,
I have cloned and experienced the same problems. I then changed my primers and sequenced again. Each time you clone a protein, the probability of mutation sites is different. Hence, as Manuele Martinelli asked to use a high fidelity polymerase is crucial for this purpose. I would like to suggest, you can try other primer set and hope they can work out.
Also, DNA size is perfect but it has some mutation which gives u truncated protein which falls short in length.
Dear Sherif,
It is possible that you can get shorter protein after a mutation.
In your case, you are obtaining correct size of DNA sequence so it shows that most probably, your primers work well. However, the mutation can cause formation of another stop codon at the internal region of your gene so that a truncated protein can be expressed even you have correct size of DNA.
To be ensure, sequencing of mutated DNA can show definite reason of this problem.
This is really annoying but I hope you will tackle this trouble.
Dear All
Thanks for your answers
I checked the sequence of the mutants and it appears to me that surprisingly, there are changes in the sequence other than the desired mutations which is very strange to me and I don't know the reason for this. what could be the mistake I have done that caused that undesired changes
These changes come depending upon the
a. Polymerase: Does it have proof-reading ability!
b. the more you probe it to UV light when you cut the band from the gel. More you contact with UV, more chances for it to be mutated.
c. PCR cycle: it depends upon the PCR cycle. Maximum cycle can also lead to mutation.
But you dont need to worry. Everybody faces such things when you construct the clone. You need to repeat the PCR and check again for sequencing. I am sure you will find one.
P.S. You can select 4-5 clone after transformation. Not every clone is mutated.
Good luck.
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