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CloningCloning Methods

Di- or multi-cistronic Cloning

It is possible to co-express two or more proteins from the same vector. There are two possible strategies:

  • The proteins are expressed under the regulation of different promoters. In this case, the genes are cloned sequentially into a vector containing different multiple cloning sites, e.g. pFastBac DUAL (Life Technologies), a baculovirus expression vector (see figure below).

  • The proteins are cloned under the regulation of one promoter. In this case, the genes are cloned into a normal expression vector containing one promoter and one multiple cloning site (di- or multi-cistronic cloning). Each gene should have its own Shine-Dalgarno sequence and start codon.

For the dicistronic cloning into a pET vector, we have developed the procedure shown in the following cartoon. This procedure can be extended for multi-cistronic cloning.

The order in which the genes are cloned into the vector can be important for the expression levels of the different proteins.