Dear Pratibha.

do you mean intracellular expression or secretion into the culture medium?

To provide more reilable suggestions i need to know more details regarding your protein and the specific mutation, however from your short description it seems that the mutations destabilize the protein folding in some way.

ciao

Manuele

What percent of dead cells you found for WT as compared to the mutants ? If you are sure that mutants are correctly assembled or you used same wild type construct to make mutants and later sequence confirmed those then most likely your proteins are not folded properly.

Dear Dr Manuele,

I used CaCl2 mediated transfection utilizing Negative and Positive Control and my three recombinant constructs. I made my first wild-type protein in pShuttle with a C-ter FLAG tagging. I used it as a template for my next mutant protein with a point mutation (which is a Knockdown resistant construct). The next mutant is made utilizing the second construct as a template with another point mutation for its activity mutant.

Thanking you for helping me in this.

Dear Dr Mahesh

The percentage of dead cells were almost same as that of other and PC. I have not done sequencing of the construct but expected to be correct by the digestion fallout. Thanking you.

Pratibha

Dear Pratibha

so your protein is a membrane protein?

i suggest you to:

1) check the sequences because mutagenesis is simple but some time tricky, just a single base frameshift due to incorrect primer annealing can cause introduction of unwanteed stop codon and i'm not sure that it will change a lot e results of a digestion check.

2) i'm nor expert in knockdown resistance mutation and so i do not. know if it is known at is affect the protein structural stability but probably you can try to perfom the second mutation in the wt construct instead that the second to check if at least this mutant it expreess.

good luck

Manuele

My protein is mainly a nuclear protein and some limited functions has been known as a cytoplasmic protein.

I have introduced a point mutation but keeping the same amino acid (just changed some nucleotides based on its ambiguous nature). But I guess (based on your answer) still it has some serious folding issues which lead to its failed expression. Thank you.

Sometimes mutations make the constructs toxic to cells. I have seen that for many of my mutants. But if your percentage of dead cells did not change, then most commonly, this is due protein misfolding.

Dear Prathiba

theoretically a point mutation that keeping the aminoacid cannot affect protein folding, if it is located in the n term of you gene can change a little the Rna conformation and then the speed rate of protein production but it is strange that it induce a so serius folding issue. check the sequences to be sure what really happened during your mutagenesis.

ciao

Manuele

Thank you so much for your help. I have finally considered as some non specific mutation occured in my construct as the protocol to make mutant construct involves 60 cycles of PCR. So I am preparing the construct again. I will confirm about its success. Thanking you all!

Dear Pratibha.

i dont know which kit or enzime are you using fot mutant generation but using a good polinerase as kapq hifi, clone amp, q5 you can obtain nice results with 30 cicles.

and i reccomend to you to check the final sequence of your clone, i know that it is expensive but you can save money performing mutagenesys using pipe cloning instead a commercial single point mutation kit and use it for sequencing that is essential when you performing mutagenesis . if you are interested to know more detail you can find more details here

https://www.researchgate.net/publication/317411668_PIPE_Polimerase_incomplete_primer_extention_method_for_enzyme_free_rapid_gene_cloning_deletion_insertion_and_mutagenesis

good luck

Manuele

Dr Manuelle

Generating the mutant construct involves two cycles of PCR . first for the two fragments and second for the full length amplification. This includes 30+30=60 cycles.

I use Thermo LA Taq Enzyme for the PCR.

Hi Pratibha,

60 cycles with a non-proofreading polymerase could definitely be the issue.

To introduce a point mutation you can use the QuikChange protocol:

https://www.agilent.com/cs/library/usermanuals/Public/200523.pdf

The kit is expensive, but you can follow the protocol with your own reagents. 16 cycles with a proofreading polymerase (we use Pfu) works well for us.

Thank you Dr Philip for your help.

To generate the desired mutant construct I need to perform 2 PCR cycle; one-by-one. And I use Thermo Taq Polymerase which has a proof reading ability and the kit is expensive as well. I am trying another time. I will inform here about my results.

Dear Pratibha

i do not undertand why for introduce a single point mutation you need to perform 2 sequential pcr. generally 1 is enough..if you share the sequence in private. maybe i can help you to review you primers and reduce the pcr cicle to 1.

manuele

Hi again Pratibha,

Why do you need to do 2 PCRs to make a single mutation? You should only need one. Can you share the protocol you are following?

You can follow the QuikChange protocol without actually buying the kit.

I also think Taq generally does not have proofreading ability unless you are using some recombinant version with proofreading engineered in. I would always use Pfu for mutagenesis. Promega has a nice explanation of the difference here:

https://www.promega.co.uk/resources/pubhub/enotes/why-select-pfu-dna-polymerase-over-taq-dna-polymerase/

Hi,

If you use SDM method then one primer set with desired mutation and one round of PCR is enough to creat point mutation.I feel you can use Q5 from NEB which is working very good and I could make all my point mutation with desired gene. If you make the catalytic site mutation with other aminoacids some time the expression is problem try using Ala to replace your target aminoacids.

If you are using overlapping PCR then you have to amplify the target gene has two fragments and and go with final PCR.

following

Hello all,

I have repeated the previous protocol as mentioned by my lab, and I have been successful in achieving the expression of the mutant construct. The probability of mutation in my earlier construct may have led to the non-expression of the construct.

Thank you all for your help. I am heading to the cloning of the third motant using the last mutant construct as a template.

Regards,

Pratibha