Human interferon gamma: significance of the C-terminal flexible domain for its biological activity

Effect of the 3′-Terminal Truncation of the Human interferon-Gamma Gene on Plasmid Segregation inEscherichia Coli

“…Plasmids, genes and bacterial strain Expression plasmids were constructed on the basis of the cloning plasmid pBR322 in which a synthetic constitutive promoter (P 1 ) followed by a synthetic ribosome binding site (SD sequence) were substituted for the tet gene promoter (16). The series of 3'-end gradually (by 9 bp) truncated hIFNγ genes (designated hIFNγΔ1 to hIFNγΔ7) were constructed by PCR using specific primers (16).…”

“…The series of 3'-end gradually (by 9 bp) truncated hIFNγ genes (designated hIFNγΔ1 to hIFNγΔ7) were constructed by PCR using specific primers (16). The hIFNγ gene and its derivatives were cloned in HindIII/BamHI restriction sites as shown in Fig.…”

“…Protein yield and hIFNγ-mRNA determination Samples of 20 ml LB medium supplemented with 50 μg/ ml ampicillin were inoculated in a ratio of 1:50 with fresh overnight cultures of transformed E. coli LE392 cells and cultivated to a cell density of A 595 = 0.7 at 37 °C (16). the yield of recombinant hIFNγ was measured by ELISA (16) using a sequence specific anti-hIFNγ monoclonal antibody (15).…”

“…the yield of recombinant hIFNγ was measured by ELISA (16) using a sequence specific anti-hIFNγ monoclonal antibody (15). The relative content of hIFNγ-mRNAs was determined by hybridization with a 19 nt long 32 P-labeled oligonucleotide specific for the hIFNγ gene as previously described (16).…”

“…The copy-number of the plasmid pΔP 1 IFNγ was 13±1.9. The yields of recombinant protein and hIFNγ-mRNA that also can be considered as potential factors interfering with plasmid segregation have been examined earlier by Nacheva et al (16) and were compared with plasmid copy-number and the values of the model parameters (Fig. 3).…”