Human interferon
α2b (hIFN
α2b) is the most important member of the interferon family.
Escherichia coli, yeasts, mammalian cell cultures and baculovirus‐infected insect cells have been used for expressing recombinant human interferon. Recently a
Pichia pastoris‐based expression system has emerged as an attractive system for producing functional human recombinant IFN
α2b. In this regard, gene dosage is considered an important factor in obtaining the optimum expression of recombinant protein, which may vary from one protein to another. In the present study we have shown the effect of
IFNα2b gene dosage on extracellular expression of IFN
α2b recombinant protein from
P. pastoris. Constructs containing from one to five repeats of IFN
α2b‐expressing cassettes were created via an
in vitro multimerization approach.
P. pastoris host strain X‐33 was transformed using these expression cassettes. Groups of
P. pastoris clones transformed with different copies of the
IFNα2b expression cassette were screened for intrachromosomal integration. The IFN
α2b expression level of stable transformants was checked. The copy number of integrated
IFNα2b was determined by performing qPCR of genomic DNA of recombinant
P. patoris clones. It was observed that an increase in copy number generally had a positive effect on the expression level of IFN
α2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of
IFNα2b. It was also observed that an increase in drug resistance of a clone did not guarantee its high expression, as integration of a marker gene did not always correlate with integration of the gene of interest. Copyright © 2013 John Wiley & Sons, Ltd.
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