I am trying to express a receptor (G-coupled protein receptor) in the membrane of S.cerevisiae. I have cloned the gene in a pITy plasmid, but after check the sequence I discovered a mutation in the kozak sequence. The rest of the gene sequence and the tag are correct. I have also checked the mRNA and it is present in the transformed colonies. But I cannot detect protein expression by Western blot. I don't know if the problem is in the sequence or could be due to the induction conditions (galactose, temperature...) or to the lysis method for isolating the membranes. What do you think?
Mutation in kozak sequence may highly effect on protein expression in eukaryotes. You could have very weak expression, even undetectable. I suppose you should try do cloning once again.
It is possible to express proteins with partial or no kozak sequence, however in this case expression levels will be low compared to full kozak sequence. I believe that you have to trouble shoot others in your list. I am not sure about the induction conditions, but as far a doing western blots concerned, improving the lysis method for purifying membranes or using a sensitive western blot detection system would certainly improve the detection of your protein of interest. For western blots, using chemiluminesence system (Millipore or Pierce) at the final step and using X-ray films or photographic imager to collect the signals is considered as the most sensitive system. Hope this helps.
As Dawid said, kozak sequence may affect the translation to a great extent. you should consider cloning again. even if there is little protein would it be worth for you? what is the purpose? also it is good idea to rule out other reasons like i always run duplicate gels one for western analysis and other for staining.
All the best
Mutation in the Kozak sequence may affect the translation of a mRNA but cannot solely be accounted for no expression. Many genes and mRNAs lack a perfect Kozak's sequence and yet the transcript is effectively translated. Your problem is somewhere else. It can be the host strain, the vector, ill-designed or selected translocon and other factors. Try changing the host strain and the vector in a logical pattern - this will give you some answer to orient yourself.
It is always advisable to use a yeast(S.cerevisiae) optimized codon of your gene of interest . It is common problem in yeast expression system that the lack of compatible tRNAs in yeast will inhibit the translation process. You can also try to detect the protein using antibodies if available which is more sensitive(eg.ECL)and if the quantity of protein may as low as undetectable, concentrating the sample several times sometimes help us to detect the protein. The signal sequence to target the membrane may give some troubles, please make sure it is working and in correct orientation in the mRNA...ATB
To check if you have a problem with the initiation of translation (possibly because of a non functional Kozak sequence) you might heck if the mRNA is effectively transcribed. If as you wrote, you Kozak sequence is mutated, it might be an indication that it is important, even if it is not always required for efficient translation.
Hi Miriam, You will be well-advised to clone your gene target again to avoid mutations in the Kozak sequence in the first place. However, please also note that there could be additional problems on the level of protein detection due to insufficiently characterized antibodies. A substantial body of literature addresses detection problems of G-coupled proteins on protein level where commercially available antibodies fail to detect protein or turn out to be unspecific. A good starting point is an editorial article by Martin C. Michel et al. (Naunyn-Schmied Arch Pharmacol (2009) 379:385–388) where the editors refer to a set of manuscripts highlighting the lack of specificity and selectivity of antibodies against G-protein coupled proteins. Make sure that the antibodies you are using are validated.
Considering the protein that you are trying to express, the active protein may be toxic when expressed at high levels. Therefore, the mutation in the kozac sequence could be due to evolutionary pressure to decrease the expression of the enzyme. We have seen this several time in my lab when trying to express (at high levels) either membrane proteins and polyketide synthases. Are you expressing the protein constituatively? If so, and you had difficultly transforming the constructed plasmid, you may consider switching to an low expression, inducible promoter.
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