Studies to analyse the relationship between IFNα2b gene dosage and its expression, using a Pichia pastoris-based expression system.
Human interferon α2b (hIFNα2b) 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 IFNα2b. 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 IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b-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 IFNα2b 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 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. 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.
Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Mohsin Ahmad
MA
Khan
Nadia
N
Hassan
Nadeem
N
Ahmad
Muhammad Islam
MI
Khan
Ahmad Usman
AU
Zafar
Faidad
F
Khan
Tayyab
T
Husnain
D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins
Pichia pastoris; Zeocin; expression cassettes; gene dosage effect; in vitro multimerization; transformation