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Kozak consensus sequence for insect expression


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#1 Christ

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Posted 13 March 2009 - 05:08 AM

Hi,

just learned the Kozak consensus sequence (RxxAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG)) seems to be important for protein expression in eukaryotic cells, but I don't know how important it is. If we happen to have a TAAAUG start, will the followed protein be expressed in insect cells. I seached some frequently used commercial vectors such as pAcGP67A (UCUAUG) and pDEST20 (UCCAUG), they seemed not using the Kozak sequence. I was confused, should we have to include the kozak sequence in our constructs? Anyone knows ?

Hongmin

#2 klinmed

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Posted 13 March 2009 - 12:24 PM

Hi,

just learned the Kozak consensus sequence (RxxAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG)) seems to be important for protein expression in eukaryotic cells, but I don't know how important it is. If we happen to have a TAAAUG start, will the followed protein be expressed in insect cells. I seached some frequently used commercial vectors such as pAcGP67A (UCUAUG) and pDEST20 (UCCAUG), they seemed not using the Kozak sequence. I was confused, should we have to include the kozak sequence in our constructs? Anyone knows ?

Hongmin

There is little evidence to strongly indicate that a conventional Kozak sequence is a major requirement for high expression levels in baculovirus systems. One good general rule though is to keep 5´and 3´untranslated regions as short as possible.

Chang MJ et al. (Virology, 1999, 259:369-83 ) recommend a translation initiation context of NNNNNAUGAa/c/gN. In contrast, Luckow (one of the fathers of the bac system) has described that A at -3 and a pyrimidine at -1 and +5 is a common feature of bac ORFs. There is a strong preference not to have a G in the 7 nucleotides immediately preceding the AUG (O´Reilly DR, Miller, LK and Luckow VA : Baculovirus expression vectors: a laboratory manual).
From personal experience I would say that use of a standard Kozak consensus sequence does not appear problematic. However, be far more carefully at being "fancy" by blindly adding epitope tags. An N-terminal His tag will almost certainly lower expression, an N-terminal FLAG may result in no expression at all (at least for a secreted protein).

This is a very good question, unfortunately without a good answer.

Edited by klinmed, 13 March 2009 - 12:53 PM.





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