Applications for StarGate Cloning
Protein:Protein Interaction (PPI) Analysis
The Twin-Strep-tag®:Strep-Tactin® system provides optimal solutions for protein:protein-interaction analysis.
Twin-Strep-tag® vectors for bait cloning
The StarGate cloning system is a perfect tool for efficient screening and fast identification of the optimal tag for protein:protein interaction (PPI) investigation with a given bait. Once the bait protein is cloned into the Donor Vector, a large selection of Acceptor Vectors for its expression with different tag arrangements is available.
For the different PPI analysis methods we provide Acceptor Vectors equipped with the following features:
|PPI analysis methods||Host||Tag||Terminus||Vector|
|Mammalia||C-terminus||p-DSG-IBA 103/ pESG-IBA 103|
|N-terminus||pDSG-IBA 105/ pESG-IBA105|
Protein:protein-interactions (PPI) govern almost all important processes in living organisms. Thus, their rapid and accurate determination and investigation is a major challenge in life sciences.
Based on its Twin-Strep-tag®/Strep-Tactin® system IBA provides optimal solutions with its different determination systems for protein:protein-interaction analysis based on the procedure described in Rigaut et al., 1999.
"Protein Complex Analysis combining a one-step co-purification with a mass spectrometry approach"
Rigaut et al., 1999
- A tagged bait protein is expressed in the target cell. At a given time point, cells are harvested and lysed.
- The lysate containing the bait with putatively interacting preys is subjected to tag-based affinity chromatography.
- Isolated protein complexes are analyzed by SDS-PAGE and silver staining, and compared to mock isolates.
- Potential preys could be identified by mass spectrometry or verified by Western blots with specific antibodies.
StarGate provides a simple cloning procedure for a multitude of expression vectors
Time saving, easy to handle and efficient one-tube cloning
With StarGate we developed a cloning system which allows a fast and convenient screening for the optimal expression features. Rapid and standardized subcloning of an arbitrary gene into a wide collection of expression vectors (Acceptor Vectors) is enabled without time consuming planning.
The bacterial 14 kDa-protein “azurin” was cloned and expressed by means of 21 different E. coli specific StarGate® Acceptor Vectors. Comparable amounts of E. coli cells were harvested 3 hours after induction of protein expression with anhydrotetracycline in case of pASG vectors containing the tightly controlled tet-promoter and with IPTG in case of pPSG vectors, containing the T7-promoter. Then, cell samples were lysed with gel loading buffer, heated for 5 min to 95 °C and then subjected to 15 % SDS-PAGE with subsequent Coomassie staining. Periplasmic secretion by means of ompA led in all cases to accumulation of comparable amounts of the protein of interest (lanes 11-15). This could be expected as azurin is also secreted in its authentic host P. aeruginosa. In case of cytosolic expression, however, interesting aspects became obvious since it was not expected that cytosolic expression of azurin is possible at all. Expression was enabled in case of N-terminal truncation by fusion of an affinity tag and tightly controlled expression of the tet promoter (lanes 2,3,7,8 and 9). This may be e.g. explained by some unusual secondary structure of the mRNA when the sequence encoding the mature protein is brought into close proximity to the 5’ untranslated region containing the Shine/Dalgarno sequence which leads to poor initiation of translation. Further, in case of small tags having only low influence of azurin folding/degradation, the use of the tightly regulated tet-promoter seems to be clearly preferable over the strong T7 promoter (compare lanes 2 and 3 with lanes 17 and 18 where the same protein construct is expressed with different success). Leaky expression of azurin in case of pPSG vectors may exert some toxic effect leading to counter selection and plasmid loss prior to induction of expression. In case of fusion of GST, however, the strong T7 promoter provides a higher level of expression (compare lane 4 with lane 19). This may be explained by the presence of the larger fusion partner GST. GST is in contrast to a peptide tag an efficiently folding, stable protein which obviously overcomes the adverse characteristics of the smaller protein “azurin” so that the stronger T7 promoter becomes effective in this case - in spite of poor regulation. With this comparison, the authors want in no way favour or downgrade any of the shown expression and/or purification systems as the situation may be completely different for another protein of interest (data not shown). It is only to show that initial screening for expression conditions may be worthwhile and may lead to unexpected results.