LIC insertion for dual protein expression

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LIC insertion for dual protein expression

Post by cpmf752004 » Mon May 23, 2011 7:21 pm

Dear All,

I currently have a gene coding for a two domain protein cloned into an expression vector (pET28b). I would like to express the two domains as separate genes but in the same vector.

I though that I could do it by PCR using two primers, one 5'-3' that anneals with the start of the second domain and that contains a LIC compatible 5' sequence. The other 3'-5' primer anneals with the end of the first domain and also contains a LIC compatible sequence on its 3' end. I will obtain then a linearized vector that, after treatment with T4 Pol+dATP I can anneal with a pETDuet LIC adaptor that will provide the T7 promoter, RBS, etc. for the expression of the second domain.

As I am not a molecular biology expert I will appreciate the opinion of the forum about if this strategy is a reasonable one to try.



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