- Poster Presentation
- Open Access
Human Prolactin (hPRL) and Growth Hormone (hGH) distinct behavior under bacteriophage lambda PL promoter control
© Soares et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Nalidixic Acid
- Repressor Protein
- Secretion Level
- Mammalian Expression System
When producing recombinant protein in E. coli for therapeutic use, it is desirable not only to obtain substantial amounts of it, but also make sure that potential contaminants such as antibiotics or inducing agents (isopropyl-beta-D-thiogalactopyranoside, IPTG or nalidixic acid) will not taint the final product. To prevent this shortcoming we can use expression systems where the promoter is activated by temperature shift, which denatures the controlling repressor protein cIts, allowing promoter activity. While in our hands hGH was successfully expressed and secreted in E. coli periplasm with yields in general well above 1 μg/mL/A600, after a temperature shift from 30°C to 42°C , attempts to express a related protein hormone (hPRL) with basis on the same protocol were not successful, providing 0.03 μg/mL/A600 at the most. Knowing that hPRL compared with hGH is a much more labile protein, we tried to obtain it from the same strain, but without the presence of the repressor protein and under optimized temperature conditions.
hGH periplasmic secretion level activating at different temperature and utilizing hGH-secreting W3110 strains, with or without the repressor gene (cIts).
phGH-DsbA-λPL+ pRK-248 cIts (μg/mL/A600 ± SD)
phGH-DsbA-λPL(μg/mL/A600 ± SD)
1.0 ± 0.14 (n = 2)
1,31 ± 0.38 (n = 4)
1.61 ± 0.11 (n = 3)
hPRL periplasmic secretion level in E. coli W3110, at different temperatures utilizing a vector containing (phPRL-DsbA-cIts -λPL) and one not containing (phPRL-DsbA-λPL) the repressor gene.
phPRL-DsbA-λPL+ pRK-248 cIts (μg/mL/A600 ± SD)
Statistical significance a
0.001 (n = 1)
0.14 ± 0.02 (n = 6)
0.73 ± 0.07 (n = 5)
P < 0.001
0.03 (n = 1)
0.92 ± 0.10 (n = 6)
P < 0.01
0.60 ± 0.12 (n = 8)
P < 0.001
0.02 (n = 1)
0.19 ± 0.05 (n = 4)
P < 0.001
Since it has been reported that the lack of repressor could easily lead to plasmid loss , a study was carried out determining hPRL periplasmic yield in the strain lacking cIts after two growth periods corresponding to 10 and 50 E. coli generations, obtaining 0.64 ± 0.05 and 0.78 ± 0.03 μg hPRL/mL/A600 respectively. Also the presence or not of antibiotic (amp) did not influence the specific expression yield for at least 40 generations.
This same strain is being utilized for setting up a rapid and flexible feed batch fermentation in a laboratory bioreactor, obtaining up to now ~7 μg hPRL/mL with an optical density of 42.4 A600.
A relatively high hPRL periplasmic secretion (up to 0.9 μg/mL/A600), never reported before, has been obtained by constitutive expression of the unrepressed λPLpromoter, at 37°C. The expression level is approximately 10-fold higher than that obtained in previous work  by using an IPTG-activated tac promoter. We can conclude that these data open the way to the utilization of E. coli instead of insect or mammalian expression systems for the production of an authentic and highly homogeneous hPRL.
Supported by FAPESP and CNPq.
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