Cloning

The Direct Transfer Cloning procedure is recommended in cases the optimal expression and purification system is already known. In this procedure, a PCR product containing the gene of interest can be directly inserted into the appropriate Expression vector (Acceptor Vector).

In the first step, the gene of interest (GOI) will be equipped at both termini with combinatorial sites (orange and red) and the Esp3I recognition sites (light blue), which are important for oriented insertion of the PCR fragment. This PCR product is in the second step integrated into the appropriate Expression vector resulting in the final expression construct, the so called Destination Vector. The formation of the correct Destination Vector is monitored via blue-white screening on LB-Agar plates containing X-Gal. used for transformation or transfection of the expression host.

Step 1: Entry Cloning for Donor Vector generation

In the first step, the gene of interest (GOI) will be equipped at both termini with combinatorial sites and the LguI recognition sites, which are important for oriented insertion of the PCR fragment into pENTRY-IBA51. This is done by PCR using a proofreading polymerase.

Recombination of the PCR product with the Entry Vector at the combinatorial sites (red and orange) leads to generation of the Donor Vector. This step involves loss of the LguI restriction sites (dark orange with arrowheads), making the recombination reaction unidirectional and thereby highly efficient.

In the resulting Donor Vector the same combinatorial sites are now flanked by the Esp3I recognition sites (light blue), thereby enabling a highly efficient and specific StarGate® gene transfer process into Acceptor Vectors in a similar manner.

Step 2: Transfer Cloning (Destination Vector Generation)

In the transfer reaction step, the GOI is transferred from the Donor Vector into an Expression vector (Acceptor Vector). The Acceptor Vectors provide the desired genetic surroundings (i.e. affinity tag, promoter (prom), additional signal sequences, etc.). In cases the optimal expression and purification conditions of the GOI are unknown, a variety of different Acceptor Vectors should be chosen.

The transfer from the Donor Vector into the Acceptor Vector is assisted by Esp3I as restriction enzyme and T4 DNA Ligase. In the resulting Destination Vector, the GOI is placed under control of the selected promoter (prom), allowing GOI expression in the selected expression host. In this example, an affinity tag (green) is fused to the C-terminal end of the GOI expression product.

The coding sequence of azurin, a 14 kDa bacterial protein, was cloned into 10 different pASG-IBA vectors and expressed in E. coli. Comparable amounts of E. coli cells were harvested 3 hours after induction with anhydrotetracycline. Samples were lysed by boiling for 5 min at 95 °C with gel loading buffer and analyzed by SDS-PAGE. Subsequently, the gel was stained with Coomassie. Periplasmic secretion by means of ompA led in all cases to accumulation of comparable amounts of the protein of interest (lanes 6-10). 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 anticipated that cytosolic expression of azurin is possible at all. Expression was enabled by fusion of an N-terminal affinity tag (lanes 2, 4, and 5), while N-terminal untagged variants did not result in expression (lane 1 and 3). The examples show that initial screening for expression conditions may be worthwhile since different constructs can lead to various results depending on the target protein properties.