title: Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product.
abstract: The aim of this research was to obtain recombinant human interferon alpha 2b (rhIFNalpha2b) from a synthetic open reading frame (ORF) overexpressed in Escherichia coli. For gene assembly, oligonucleotides were designed by Thermodynamically Balanced Inside Out (TBIO) method using the published synthetic codon optimized hIFNalpha2b ORF for high expression in E. coli. The synthetic ORF was assembled by a two-step Polymerase Chain Reaction (PCR) and cloned into a pGEM-T vector. The two-step PCR resulted in a DNA band of 522 base pairs (bp) corresponding to the size of hIFNalpha2b ORF. Fifteen recombinant pGEM-Ts were obtained and the sequencing results showed that the ORFs contained one to ten mutations with an error rate of 8.3 per kilo base. An ORF carrying one mutation was cloned into a pET32b vector and site-directed mutagenesis was performed to correct the mutation. The hIFNalpha2b ORF was overexpressed as a thioredoxin-his-tag fusion protein in E. coli BL21. The rhIFNalpha2b fusion protein was isolated from inclusion bodies (IB), renatured, and purified using Nickel columns, and all steps were monitored by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). A rhIFNalpha2b fusion protein of 37kDa in size was produced in high expression levels relative to total protein, renatured and purified from IB with a yield of 3.46mg/l without any further optimization. The purified rhIFNalpha2b was confirmed by peptide analysis with nano-LC-MS/MS2 mass spectrometry. Our current research demonstrates for the first time that by using the TBIO method a synthetic ORF encoding hIFNalpha2b gene can be expressed at high levels in E. coli.
authors.affiliation: School of Pharmacy, Institut Teknologi Bandung, Ganesha 10, Bandung 40132, Indonesia. retnoningrum@indo.net.id
authors.forename: Debbie S
authors.initials: DS
authors.lastname: Retnoningrum
authors.forename: Ratih A
authors.initials: RA
authors.lastname: Ningrum
authors.forename: Yohanes N
authors.initials: YN
authors.lastname: Kurniawan
authors.forename: Ana
authors.initials: A
authors.lastname: Indrayati
authors.forename: Heni
authors.initials: H
authors.lastname: Rachmawati
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins