Stability study of the producer strain of the analgesic peptide HCRG21

The development of new, more effective analgesic drugs with high affinity for biological targets is a critical challenge. The peptide HCRG21, a TRPV1 channel blocker derived from the sea anemone Heteractis magnifica, is considered as a promising candidate for a new analgesic drug.

Aim. To investigate the genetic and expression stability of the Escherichia coli strain Origami2(K12)/pLysS/pSMT3_HCRG21, which produces the SMT3_HCRG21 fusion protein.

Materials and methods. The viability of the working seed lot (WSL) during sequential passaging was assessed by calculating the cell titer in each passage. The number of generations of WSL cells was determined based on the viable cell titer values for each passage using an appropriate equation. The expression level of the fusion protein during continuous cell passaging was calculated as the ratio of the fusion protein amount to the total cellular protein in the cell lysate. Protein content was determined using the Lowry method and electrophoresis. The size of the DNA region encoding the SMT3_HCRG21 fusion protein was determined by PCR and electrophoretic separation, and Sanger sequencing was performed to confirm its genetic identity as specified in the strain passport.

Results. During continuous passaging, the cell titer has been shown to vary from 8.3·10⁷ to 1.7·10⁹ cells/mL, and the average number of generations was 10.4 ± 2.3. Following pDNA amplification of clones from 10, 90, and 140 generations the PCR fragments of approximately 600 bp in length were obtained, which correspond to the length of the nucleotide sequence of the pSMT3_HCRG21 genetic construct (541 bp). The nucleotide sequences of the obtained PCR fragments were identical to those specified in the strain passport. It was demonstrated that the average content of the fusion protein across generations remained at 16 %, indicating a high stability of the strain productivity level.

Conclusions. The biosynthetic activity and viable cell titer of the E. coli strain Origami2(K12)/pLysS/pSMT3_HCRG21 remained stable; therefore, the strain can be used for industrial production of the analgesic peptide HCRG21.

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