Screening for the optimal induction parameters for periplasmic producing interferon-&#945 2b in Escherichia coli

Screening for optimum induction parameters to improve the production of periplasmic interferon-α2b (PrIFN-α2b) by recombinant Escherichia coli was conducted using shake flask culture. Recombinant E. coli Rosetta-gami 2(DE3) harboring the plasmid pET26b containing IFN-α2b gene under the control of the T7lac promoter was used, where the induction was accomplished by isopropyl β-D-1- thiogalactopyranoside (IPTG). The induction parameters (inducer concentration, point of induction, induction temperature and the length of induction) were analyzed to find the suitable range to be used for further optimization process. From the analysis, narrow range of induction temperature from 16 to 30°C and IPTG lower than 2 mM were found suitable for induction of PrIFN-α2b. On the other hand, early log phase was the preferred time to initiate the induction and the length of induction was dependent on the combination of other induction parameters used.Key words: Interferon-2b (IFN-2b), induction parameter, Escherichia coli, periplasm, shake flask culture

Effect of promoter strength and signal sequence on the periplasmic expression of human interferon- &#9452b in Escherichia coli

Two plasmids, pFLAG-ATS and pET 26b(+), were studied for the periplasmic expression of recombinant human interferon-2b (IFN-2b) in Escherichia coli. The pFLAG-ATS contains ompA signal sequence and tac promoter while pET 26b(+) contains pelB signal sequence and T7lac promoter. It was observedthat periplasmic expression of IFN-2b from pET 26b(+) was around 3000 times higher than pFLAGATS. Difference in the expression level was attributed to the difference in the promoters and the signal sequences. In silico analysis of mRNA secondary structures were analyzed using Vienna RNA packageand MFOLD. The results suggested that the increase of expression would mainly due to the difference in the translation initiation associated with secondary structure of mRNA transcribed by both plasmids