سال انتشار: ۱۳۸۴
محل انتشار: چهارمین همایش ملی بیوتکنولوژی ایران
تعداد صفحات: ۴
Fariba Ataei – Institute for Genetic Engineering and Biotechnology.
Alireza Zomorodipour – National Institute for Genetic Engineering and Biotechnology.
Hossein Ghanbarian – Drug Applied Research Center, Research & development Complex, Tabriz University of Medical Sciences, Tabriz-Iran
Bagher Yakhchali – Drug Applied Research Center, Research & development Complex, Tabriz University of Medical Sciences, Tabriz-Iran
In order to express human granulocyte-macrophage colony stimulating factor (hGM-CSF) under heat shock, two expression plasmids was constructed based on pBC(SK) plasmid. The expression cassettes in the two plasmids are equipped with a 75 base pair fragment, derived from the PL promoter of the bacteriophage lambda (_). The plasmids also contain a temperature mutant of repressor coding gene (CI857) 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. The protein analysis of the recombinant bacteria, containing either of the two plasmids, indicates in a successful expression and complete processing of the hGM-CSF precursor, following the heat shock activation of the _PL 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 plasmid. The heat-regulated plasmids, designed in this work, have provided suitable tools to study the expression of recombinant proteins under temperature up-shift in Escherichia coli, when use of chemical inducers are not desirable.