Root > Products > StarGate Expression Vectors > "Two-step-cloning" via pENTRY > Fusion Cloning > Generation of fusion proteins or artificial operons for multiple gene expression using the StarGate Fusion Cloning System

Generate fusion proteins or construct artificial operons for multiple gene expression in bacterial or mammalian cells

Fuse genes from any Donor Vector in any combination to make single fusion proteins or even to construct artificial operons for multiple gene expression in bacterial or mammalian cells. The StarGate® Fusion Cloning system allows easy and fast fusion of two different genes of interest (GOI-1 and GOI-2) by an intergenic region (IR). Only two sequential StarGate® subcloning reactions with dedicated Fusion Vectors are required. The procedure can be repeated to fuse more than two GOI´s.

 

In the first reaction, the two GOIs need to be transferred into special fusion vectors by two separate transfer reactions performed in parallel. The GOI that is intended to be positioned upstream in the final construct is cloned into a pNFUSE-IBA derivative, carrying the IR, while the second GOI is placed into pCFUSE-IBA11, which is always the same irrespective of the desired IR. The cloning into the fusion vectors is driven by the Esp3I restriction enzyme.

In the second reaction, GOI1 and GOI2 cloned in the Fusion Vectors are assembled in a directed manner using LguI as restriction enzyme into pENTRY-IBA51 by a second one-tube reaction.

Intergenic regions for tandem gene expression

The StarGate® Fusion Cloning system allows easy and fast fusion of two different genes of interest (GOI-1 and GOI-2) by an intergenic region (IR). Such an intergenic region, e.g. codes for an amino acid linker sequence directly connecting two proteins (LINK11/12). Alternatively, it may include a Shine Dalgarno (SD) or an IRES site for the construction of synthetic operons in bacterial or mammalian cells, respectively. This allows independent expression of both proteins, whereas the linker sequence produces a large fusion protein of both GOIs.

 

For detailed informations on the „intergenic regions (IR)” have a look on technical informations.

Overview on the Fusion Cloning Procedure

 

Fusion Cloning Procedure:

StarGate® fusion cloning provides the possibility to express two or more GOIs from only one Acceptor Vector. The GOIs, which are present in respective Donor Vectors are transferred to the pNFUSE-IBA and the pCFUSE-IBA vectors. The GOI, which will be located upstream, needs to be conveyed to the pNFUSE-IBA vector, this vector contains the intergenic region. The downstream located GOI is cloned into pCFUSE-IBA. The transfer is unidirectional and oriented insertion of the gene is ensured.

In a next step, both genes are assembled in the Entry Vector using LguI restriction enzyme.

The resulting Donor Vector provides again Esp3I recognition sites for the transfer of the expression cassette (containing both GOIs) into the Acceptor Vector.