Dear Emile,

In general, Kozak sequence is required, for the Kozak sequence (A/GCCAUGG) enhances translation from the correct initiation codon. While, the common elements in plasmid vector, such as CMV promoter and SV40 polyA signal may help enhance the translation efficiency of target gene, in case Kozak sequence is not present.

Genemedi got a rich experience in protein over expression and subsequent virus packaging, such as lentivirus packaging, you could find more information on www.genemedi.net/i/lentivirus-packaging

I guess Kozak sequence is necessary in your case. Otherwise expression level may not be proper.

I guess CMV promoter (a strong promoter) + the enhanced stability of mRNA for the presence of SV40 polyA signal both part of the pcDNA3.1 plasmid will help strongly the expression and translation rate of your CDS even in case Kozak sequence is not present.

If for some reason you cannot clone this sequence upstream to your ATG you could try to express your protein anyway. 

The Kozak sequence (A/GCCAUGG) enhances translation from the correct initiation codon. I suggest to include the sequence in your cloning primer.

The kozak sequence in mammalian mRNA acts as a enhancer for translation. it is required but not necessary. As said by Paolo, the CMV promoter will drive a strong expression. If you have the clone ready without the Kozak, just give a try. If it does not work then introduce the 5' kozak sequence in the same vector using Q5 kit from invitrogen or by direct methods.

Thanks

I think Kozak sequence is must in this regard. Without it the expression level might be uncleared

Hello Emile, I know I ask a long time after your question but did you get any answer so far ? I produced lentiviruses without Kozak sequence and didn't get any expression, I'm wondering if this might be the cause.

Thanks :)

Hi Vincent,

I have experience with this issue. And the answer would be YES, Kozak sequence play an important role in the initiation of the translation process. At first I got very low protein expression ( but if you're working with a strong promoter such as CMV- Actin, it's fine), then I tried to add Kozak sequence and got an improved protein yield. So I would suggest you to try putting an N-ter kozak sequence in your construct. There may be other reasons associated with lentiviral cloning which I'm not good at, but at first you can try with Kozak and see how you would go from there.

Good luck!

Hi All,

Huong Nguyen

I am having probably similar problem with my protein expression using retrovirus mediated system. My vector's have the following sequence around the Start Codon (ATG).

1. tccgccATGa

2. cccaccATGg

These proteins are intended to express under retroviral LTR promoters. Could you confirm if this sequence is sufficient for proper translation of my protein.

Vincent Turon-Lagot

I would like to know how you troubleshooted your problem with lentivirus mediated expression.

Thank you in advance.

Hi Arun Kumar B ,

I think the issue I had was due to the presence of two different ATG in the HA-tag sequence I used. The Kozac sequence didn't solve the problem. I haven't been further in those experiments yet but I'll let you know if the expression problem is solved with a new plasmid I ordered with a correct Kozac sequence and only one ATG in the HA-tag sequence.

Hi Arun Kumar B ,

Sorry for my late response; I didn't check the page so far.

I haven't used viral vector but for a non-viral one, the kozak sequence I use is: gccgccaccATG. I have found other viral vectors in publication used that same sequence. Maybe you can try that and please let me know how it works.

Good luck!

Thanks Vincent Turon-Lagot and Huong Nguyen ; I am now trying to manipulate my vectors for proper KOZAK sequences, Hope the problem will be solved.

Dear Emile,

In general, Kozak sequence is required, for the Kozak sequence (A/GCCAUGG) enhances translation from the correct initiation codon. While, the common elements in plasmid vector, such as CMV promoter and SV40 polyA signal may help enhance the translation efficiency of target gene, in case Kozak sequence is not present.

Genemedi got a rich experience in protein over expression and subsequent virus packaging, such as lentivirus packaging, you could find more information on www.genemedi.net/i/lentivirus-packaging

My personal believe is that the "Kozak" sequence is a bit of a myth. It was originally based an very few sequences. However, when we cloned new cDNAs in the eighties and you wanted to publish it, you always wanted to write something about it. So it usually went "the ATG is preceded by a Kozak consensus sequence" or "a Kozak consensus sequence hasn't been identified". Either way, the paper was cited propagating the consensus sequence. I don't know if anyone actually did a systematic study around it in recent years using the same expression system and the same gene. Having all said, it can't hurt to add A/GCC before the ATG. For the G after this you can't do much if there is an A,T, or C or you change the amino acid. If you use a TAG, it can be an advantage to eliminate the original ATG to prevent the chance of secondary initiation. Regarding the protein level itself, it probably depends more on half-life for proteins than the translation initiation efficiency and any toxic side effects of overexpression.

Hi all,

Rolf-Peter Ryseck , this is a bold statement, yet a very interesting one. I'd like to take your point on board and agree that Kozak sequence is not as strong a consensus as the Shine-Dalgarno (SD) is. But let's consider that Kozak might be some remnant of SD in eukaryotes. It would make sense that the 40S unit of the ribosome scanning the mRNA for the AUG would need more than the start codon to trigger the elongation of the translation. Some have investigated base-pairing complementarity between Kozak and 40S sequences with limited success (not unsurprisingly given the weakness of the consensus and the variety of 40S sequences). It has been postulated however that Kozak interaction with 40S could change the conformation of 40S by a mechanism of Competitive‐Displacement (

https://doi.org/10.1016/0014-5793(91)81437-D). I like this idea because it sounds like a way to stall the initiation complex, long enough so that it is dislocated and allows the large subunit of the ribosome to join.

I would not say that Kozak is an ON/OFF switch, obviously protein expression can occur without it. But it is possibly a Tuning Switch, that may improve translation. Let's not forget that many of us are introducing un-physiological over-expression in cells using strong ubiquist viral based artificial promoters. In such context, suboptimal translation might be compensated by hyper boosted transcription (and may be luckily so).

To conclude, I would recommend always including a Kozak sequence around the AUG when planning a protein expression in eukaryotes:

- because that will do no harm

- especially when you plan to use physiological endogenous promoters

- if possible by looking for the best consensus for your system (yeast, plant, mammals etc...) so the Kozak may fit better to the 40S sequence of your biological system.

Hi Arun Kumar B !

I finally got this answer on why my protein was not expressed and the problem came from the HA-Tag in N-term. By changing it to a FLAG in C-term position, I could have a very nice expression of my protein (with a Kozak sequence). Didn't think of that at first, but I think that's what solved my problem.

I wonder you may need these protocols of AAV, lentivirus and adenovirus to know the elements necessary in transfection.