Is a Kozak sequence necessary in Sindbis replicon vector?

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Is a Kozak sequence necessary in Sindbis replicon vector?

Postby Tirptz » Jul 24 2017 10:45 am

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?
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Re: Is a Kozak sequence necessary in Sindbis replicon vector

Postby relaxin » Jul 24 2017 8:13 pm

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.
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Re: Is a Kozak sequence necessary in Sindbis replicon vector

Postby Tirptz » Jul 25 2017 2:45 pm

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.
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Re: Is a Kozak sequence necessary in Sindbis replicon vector

Postby r.rosati » Jul 26 2017 8:56 am

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".
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Re: Is a Kozak sequence necessary in Sindbis replicon vector

Postby Tirptz » Jul 27 2017 7:46 am

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
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