Is a Kozak sequence necessary in Sindbis replicon vector?

Go to the profile of tirptz
Jul 24, 2017

I amplified a viral gene by PCR and cloned it into a Sindbis virus replicon plasmid vector.The vector has a human cytomegalovirus (hCMV) immediate-early promoter/enhancer element. The total plasmid side is 16kb. There is no kozak sequence before the viral gene start codon. I have been electroporating in HEK and BHK cells but the level of protein expresson is very low, around 5%. Could it be that the low level of protein expression is because of the absence of the kozak sequence? Do i need to incoporate it inorder to improve protein expression levels or what else can i do improve protein expression?


Go to the profile of relaxin
relaxin 8 months ago

Yes, a Kozak sequence is needed for efficient translation of viral RNA in eukaryotic cells. However, your low expression level may also be due to low efficiency of electroporation.

Go to the profile of tirptz
tirptz 8 months ago

I cannot rule out low transfection efficiency though i have tried a number of electroporation parameters including those recommended by the kit's manufacturer. The positive control (GFP) is being expressed well, about 90% efficiency. It is in a smaller plasmid, 7kb.

Go to the profile of Roberto Rosati
Roberto Rosati 8 months ago

If you're using pSinRep5, page 14 of the manual states "It is recommended to include a Kozak consensus sequence in your insert for proper initiation of translation of your gene (Kozak, 1990) and a stop codon for termination of the recombinant protein".

Go to the profile of tirptz
tirptz 8 months ago

I am using a plasmid assembled in the lab. Its not pSinRep5 but i guess the same principle applies. I will include a kozak sequence and compare protein expression levels. Thank you