Applications Enabled by Gateway Cloning

Common baculoviral expression systems require the use of a bacterial transfer vector to transfer an ORF of interest into the bacuiovirus genome by homologous recombination in bacteria. This requires that the reaction be carried out in E. coli cells that have fully functional recombination machinery (this is disabled in standard 

The generation of soluble, active protein is now more important than ever with the increased emphasis on expression of proteins for structural analysis. One class of proteins that has gained importance as druggable targets are human membrane proteins. Currently, baculoviral expression is one of the best avenues for the generation of soluble membrane proteins. Therefore, easier access to baculovirus expres- 

Panel B). For example, by reversing the standard orientation of the attPl site (to an attPlr site), a BP recombination with an attBl site (in reverse orientation, attBlr) results in the formation of an attRl site instead of an attLl site. This concept is key to the elegance of the Multisite Gateway system. 

Creation of Entry Vectors and Three-fragment Multisite Assembly Reaction 

Modular assembly of expression constructs including promoters, ORFs, epitope and purification tags, can now be achieved in HTP, without the use of restriction enzymes and ligase. Once a set of entry vectors has been created, the elements can be mixed and matched in an LR reaction. Elements in a multi-fragment constract can be selectively removed via a specific BP recombination reaction, thereby creating an intermediate destination vector. For example, an enzymatic pathway can be assembled using modular promoters and ORFs to create a synthetic operon. Once the wild-type activity is established, a library of mutants can easily replace specific 

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