Publication Date:
2013-11-10
Description:
Human interferon alpha-2b (hIFNα2b) is the most important member of interferon family. Escherichia coli , yeast, mammalian cell cultures and baculovirus infected insect cells have been used for expressing recombinant human interferon. Recently Pichia pastoris based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered, an important aspect to get the optimum expression of recombinant protein which may vary from one protein to another. In present study, we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris . Constructs containing from one to five repeats of IFNα2b expressing cassettes were created via in vitro multimerization approach. P. pastoris host strain X-33 was transformed with these expression cassettes. Groups of P. pastoris clones transformed with different copies of IFNα2b expression cassette were screened for intrachromosomal integration. IFNα2b expression level of stable transformants was checked. Copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that increase in copy number generally have positive effect on expression level of IFNα2b 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 . This was also observed that increase in drug resistance of a clone not guarantee its high expression, as integration of marker gene do not always correlate with integration of gene of interest. This article is protected by copyright. All rights reserved.
Print ISSN:
0749-503X
Electronic ISSN:
1097-0061
Topics:
Biology
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