Construction of an Escherichia coli-specific heat-inducible expression plasmid

Purpose: In order to express human granulocyte-macrophage colony stimulating factor (hGM-CSF) under heat shock. Materials and Methods: Two expression plasmids were constructed based on pBC(SK) plasmid. The expression cassettes in the two plasmids are equipped with a ۷۵ base pair fragment, derived from the PL promoter of the bacteriophage lambda (λ). The plasmids also contain a temperature mutant of repressor coding gene (CI۸۵۷) to regulate the promoter activity. The two plasmids differ from each other in having a transcription termination signal or not, down stream to the recombinant gene in the expression cassette. A pelB signal sequence was also used in order to have the recombinant protein in the periplasmic space of Escherichia coli. The efficiency of the constructed plasmids was demonstrated by heat-regulated expression of hGM-CSF. Results and Discussion: The protein analysis of the recombinant bacteria, containing either of the two plasmids, indicates a successful expression and complete processing of the hGM-CSF precursor, following the heat shock activation of the λPL promoter. In order to enhance the applicability of the terminator containing plasmid, for the expression of other proteins of interest by heat regulation, a multiple cloning site including eleven unique restriction sites was inserted in the plasmids. The heatregulated plasmids, designed in this work, have provided suitable tools to study the expression of recombinant proteins under temperature up-shift in Escherichia coli, when the use of chemical inducers are not desirable.

کلیدواژه ها:

Heat Shock ، &lambda ، PL Promoter ، CI۸۵۷ Gene ، Periplasmic Expression and Human Granulocyte-Macrophage Colony Stimulating Factor (hGM-CSF) ، القای حرارتی ، پروموتور PL ، ژن CI ، ترادف نشانه ، بیان پریپلاسمی و hGM-CSF