| dc.description.abstract | Recombinant bacteria are used for production of human proteins such as the medical important cytokine Interferon-alpha-2b (IFN-α-2b). In the present study, Escheriachia coli (E.coli) and Cupriavidus necator (C.necator) have been used as expression hosts for production of human IFN- α -2b. The Pm/XylS expression system, originating from the Pseudomonas putida TOL plasmid, is used for recombinant gene expression. A set of expression vectors based on the broad-host-range plasmids RK2 or pBBR1, in combination with the Pm/XylS expression cassette, was constructed. Previous research has identified high-expression variants of regulatory elements of the Pm/XylS expression cassette, combined these variants and successfully obtained high protein expression. In order to evaluate the expression of IFN- α -2b, a high-expression variant of the Pm/5'UTR element in combination with a codon-optimized version of the IFN- α -2b gene was compared with the wild-type Pm/5'UTR variant in combination with a codon-optimized IFN- α -2b gene. In addition, the expression of IFN- α -2b in the presence/absence of the signal peptide pelB was evaluated. Soluble and insoluble IFN- α -2b fractions were observed with both E.coli and C.necator as expression host.However, it was generally detected higher expression of IFN- α -2b in E.coli than C.necator. Strong expression cassette elements without pelB were favorable for the expression of soluble IFN- α -2b, while strong expression cassette elements in combination with pelB promoted the expression of insoluble IFN- α -2b. For both of the expression hosts, a larger fraction of the protein was found as insoluble than soluble IFN- α -2b. As far as one knows, detectable expression of soluble IFN- α -2b is not previously been reported in neither E.coli nor C.necator. This study had reported clearly detectable levels of soluble IFN- α -2b in both of these strains | |
