Escherichia coli is one of the most widely used hosts for production of heterologous proteins. One example of these recombinant proteins is the human growth hormone (hGH), a pituitary-derived polypeptide with a wide range of biological functions including protein synthesis, cell proliferation and therapeutic applications in different treatments.
In commercial production with recombinant microorganisms, one of the most important problems is plasmid instability, a tendency of the transformed cells to loose their engineered properties because of changes to, or loss of, plasmids. Instability of plasmids can result in a significant loss in productivity.
The plasmid stability test determines what proportion of the cells maintain the target plasmid and the ability to express the target protein.
In the present study we produce the human growth hormone (hGH) using Escherichia coli and we are particularly interested in the establishment of a protocol to study the stability of the recombinant plasmid containing the gene that codifies the human growth hormone (hGH) in pET30 a(+) vector.
In order to measure the growth rate, cells harboring plasmids were used to estimate the number of generations that were used in the experimental plots, to define a number of generations used for the analyzes.
The culture of the strain E. coli C43 (DE3) carrying the construct gene was incubated in M9 minimal medium, in bioreactor cultivation with an agitation cascade through automatic adjustment of agitation speed to maintain DO at 30%, to compare the plasmid stability in a medium with or without kanamycin. Diluted samples are plated, every generation time, on selective and non selective LB plates. The ratio of colonies on the plates represents the plasmid stability.
After 32 hours of cultivation with IPTG induction, the plasmid presented a high stability in both media, with or without kanamycin, indicating a good resistance to this antibiotic with protein expression until the end of cultivation.