Efficient procedures to generate functional recombinant proteins or protein complexes are of key importance in state-of-the-art life sciences. Many tools like various expression hosts (bacteria, yeast, insect and mammalian cells), promoters, affinity or fluorescent tags are currently available to express, purify, detect or immobilize recombinant proteins. Due to the diverse nature of proteins, however, it is impossible to predict which combination of these tools will perform best. Therefore, many have to be tried to identify the optimal solution. To systemize and accelerate this initial search which is crucial for successful subsequent proteomic research, we have developed the StarGate Cloning System. StarGate offers rapid and highly efficient subcloning of an arbitrary gene – initially cloned into a Donor Vector - to simultaneously fuse it with many different genetic surroundings via transfer into Acceptor Vectors to generate Destination Vectors. The latter enable the efficient expression of your protein with various features (e.g. different tags and different promoters) in different hosts.
Standard StarGate® procedure
Step 1: Entry reaction In a first step, the gene of interest (GOI) is equipped at both ends with combinatorial sites (4 bases) by PCR and is inserted into an Entry Vector by a simple one-tube reaction. The opened Entry Vector contributes the recognition sites and provides an operative linkage with the combinatorial sites for the highly specific StarGate® gene transfer process (step 2).
Step 2: Transfer reaction After sequence confirmation, the resulting Donor Vector is the source for the highly parallel subcloning of the GOI by a second simple one tube reaction into a multitude of Acceptor Vectors, each providing a different genetic surrounding like host specific promoters and different purification tags. The resulting Destination Vectors are then transformed into the corresponding host cells for further experiments. Acceptor Vectors are listed in the Acceptor Vector Overview Table.
Optional intermediate step: Fusion Cloning Via an optional intermediate step between entry and transfer reaction you can construct artificial operons for multiple gene expression in bacterial or mammalian cells or generate fusion proteins.
We offer an easy-to-use software, the StarPrimer D´Signer 2.0, to facilitate the design of primers for the ENTRY Vectors both for StarGate Standard Cloning and for StarGate Mutagenesis Cloning. The software guides you step-by-step through the whole PCR procedure.
