2 replies to this topic

[cpmf752004]

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 (see PDF attached) 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.

Thanks

Carlos

Attached Files

•  tb389-000.pdf   565.07K   227 downloads

[Adrian K]

I use pet46EK/LIC, cloned 13 genes so far, no major issues.

But if I remember correctly, pet28b is not a LIC vector.

Also, i really doubt whether the LIC duet vector really work for co-expression...really.

[cpmf752004]

adrian kohsf, on 23 May 2011 - 05:21 PM, said:

I use pet46EK/LIC, cloned 13 genes so far, no major issues.

But if I remember correctly, pet28b is not a LIC vector.

Also, i really doubt whether the LIC duet vector really work for co-expression...really.

Thanks Adrian.

pET28b is not LIC compatible itself but I would not need to insert the 5' and 3' ends of the two domain protein because they are already cloned in the vector. What I need is to insert a LIC DUet adaptor between the 3' end of the first domain and the 5' end of the second domain in order to express both domains simultaneously but as separated proteins.

                    
             lic-overhang                                                                                    
                         \___                        PCR                          \               LIC              \               \
5' |=====================|====================| 3'   ----->  |===========     +    ==========|   --->  |=====   +   ===        +    =======|
                     ____                                                \                                   \         \
                         \lic-overhang

          Domain 1              Domain 2                       Domain 1             Domain 2            Domain 1    LIC Duet       Domain 2
                                                                                                                      adaptor