2.4. Production and Purification of Recombinant Protein TSPphg

The highly efficient expression of TSPphg in the host E. coli BL21 cells and its rapid purification using the special thermolysis method were already described in detail in our previous publication [15]. Briefly, TSPphg gene was amplified by PCR with gene-specific primers from the phage TSP4 genome (forward: 5’-CATGCCATGGCAATGCGTCTACCGACTAAGAC-3’ and reverse: 5’-CCGCTCGAGTTTACCTCCTAGCAACTTGG-3’). The 5′ ends of forward and reverse primers contained NcoI and XhoI restriction sites (underlined), respectively. The modified primers were used to amplify the TSPphg gene for directional cloning into the expression vector pET-28a. The PCR program was performed as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 45 s, 58 °C for 30 s, and 72 °C for 90 s. E. coli BL21 cells harboring the pET-28a-TSPphg vector were then used as the host for recombinant protein expression. Lactose (1 g/L) was used for induction to overproduce the phage lysin TSPphg. After induction, cell pellets were collected by centrifuging at 12,000 g for 10 min at 4 °C, then resuspended in phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.4 mM KH₂PO₄ with a pH of 7.4. The recovery of thermostable TSPphg protein was performed by a direct heat treatment at 55 °C for 30 min to precipitate unstable host proteins. Subsequently, the samples were centrifuged at 12,000× g for 10 min to pellet debris, and filtered using 0.22-μm pore-size filters (Sartorius, Ulm, Germany). The final purified TSPphg dissolved in PBS was confirmed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, from 1 L of the host E. coli BL21 culture we could obtain approximately 79 mg of TSPphg.