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“Two-step-cloning” via pENTRY-IBA

The "Two-step cloning" approach via the Donor Vector is recommended in cases where nothing is known about the expression of the respective gene of interest. This procedure allows rapid and highly efficient subcloning of an arbitrary gene into a variety of expression vectors in parallel. In a first step the gene is cloned into the pENTRY vector to generate the Donor Vector. In a second step the gene can be transferred from this Donor Vector into Acceptor Vectors, which provide different genetic surroundings (e.g. tags, promoters and signal sequences). The resulting vector is called Destination Vector and is placed into the respective expression host.

 

StarGate® provides a simple cloning procedure for a multitude of expression vectors

- time saving, easy to handle and efficient one-tube cloning

Due to the diverse nature of proteins the requirements for the optimal expression conditions have to be evaluated for each protein individually. The most important features in this process are the expression host (E. coli, mammalia, yeast and insect cells), the promoter and the affinity tags (Strep-tag, 6xHistidine-tag, GST-tag and FLAG-tag).
 
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 expression example (right) of the 14 kDa protein (Azurin) illustrates the importance of finding the right expression features (nature of the tag its position and the promoter).
 
Screening for the best expression features in the beginning can save a lot of time for the complete protein expression project later on. For details on the figure please see below.
For details on the figure please click here!

Expression of the 14 kDa protein azurin with different tags and promoters

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.