|
The use of bacteriophage lambda PL as a constitutive promoter
Heller SR ; Oliveira TL; Ueda EK; Souza JM; Ozaki NA; Ribela MTCP; Bartolini P; Soares CRS
Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP
Centro de Biotecnologia
e-mail: crsoares@ipen.br
The most widely used promoter for large-scale production of human therapeutic proteins is the l PL. The promoting activity of l PL is switched off at low temperature (28–30oC) in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction (usually 42oC). The cIts regulator can be located either on the host chromosome, on a second plasmid pRK248 compatible with the cloning vehicle, or on the vehicle itself.
In our laboratory, using the system based on the control of λPL promoter by the pRK248, after a temperature shift from 30oC to 42oC, high expression levels of human growth hormone (hGH) were achieved, yields in general well above 1.5 µg/mL/A600. However, attempts to express a related protein hormone (hPRL) based on the same protocol were not successful, providing 0.03 m g/mL/A600 at most. Based on these results, and considering that hPRL is a thermo sensitive protein we decided to use strains lacking the pRK-248. Therefore we carried out expression studies, for each hormone, in presence or not of the repressor, cultivated under different temperatures.
Concerning the hGH results, at 42oC, no significant difference of expression was observed (p>0.15). In the presence of the repressor, the l PL promoter was almost totally repressed up to 37oC, while without the repressor, at 30oC, a quite high hGH secretion (1m g/mL/A600) was obtained. In the case of hPRL, at 37oC, utilizing the bacterial strain lacking the cIts repressor, a high hPRL secretion level, 0.92 ± 0.10 µg/mL/A600 (n=6; CV=10.4%), was observed, i.e. approximately 30-fold higher than that obtained with the equivalent strain containing the repressor gene.
Since it has been reported in the literature that the lack of repressor could easily lead to plasmid loss, a study was carried out to determine the hPRL periplasmic yield in the strain lacking cIts after two growth periods, corresponding to 10 and 50 E. coli generations. Yields respectively of 0.64 ± 0.05 and 0.78 ± 0.03 m g hPRL/mL/A600 were obtained. The presence or not of antibiotic (amp) did not influence the specific expression yield, for at least 40 generations.
We concluded that these data open the way to the utilization of l PL as a constitutive promoter for increasing the expression of thermo sensitive proteins like hPRL.
Supported by: FAPESP and CNPq.
|