i am trying to understand the reasons for the Hela cell lines not efficiently expressing my protein though my control has been showing good amount of protein. To see whether my protein is present in cells i did qPCR of control & sample and it seems that both follows same pattern(indicating transfection efficiency is good) Basically my RNA is present in the cells but is not able to make protein.
Suggestions
Komal.
Several reasons are possible, mostly linked to the species you're protein is derived from: codon usage may be off (check and eventually optimize); or possibly you've done some nasty things to your Kozaksequence (nucleotides immediately upstream of your start codon). Your cells are in good health? Proteins from heterologous origin may be toxic to cells, i.e. you select against translation...When we do transfections for protein expression we usually try a panel of cells: in addition to HeLa, mostly HEK293, HEK293T, CHO. It's not uncommon to get no or little expression in one cell line, while it's ok in the others...
If the mRNA is encoded by a gene that was delivered to cells through transfection, it is critical to make sure that the transfected gene encodes an mRNA that can be translated in a correct open reading frame in the transfected cells to translate protein. If the mRNA is expressed in transfected cells and protein is not detected, a number of factors could contribute to this outcome.
@christoph
My protein is from lower organism and trying to express in mammalian cells as they are the ones with mutant cell lines. I have checked kozak seq and its correct. My cells are in good condition.so i guess because of heterologous origin i am getting low expression but then how do i optimize codon usage.
how would i know protein toxic to cells ?
@Divaker
If that is the case then how some of my cells showing expression?
what are possible reasons for protein not being translated though mRNA is expressed.
You may have done this, but I would suggest that you do a DNAse digest of your RNA and include a no RT control in the QPCR. With transient transfection, you can easily get a signal from the transfected plasmid that is mistaken for mRNA otherwise. The only other idea is to optimize the time between transfection and making your protein samples. There is certainly a lag between mRNA and protein production and the optimal time frame for expression varies a lot between proteins. I would start with a time course like 16, 24, 48, and 72 hours.
It's also possible that your protein is being rapidly turned over. The time course idea is a good one, and you may also want to try some degradation inhibitors.
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