Elimination of an HuIFN alpha 2b readthrough species, produced in Escherichia coli, by replacing its natural translational stop signal.
When human interferon-alpha 2b (HuIFN alpha 2b) was expressed intracellularly in Escherichia coli as insoluble aggregates, a HuIFN alpha 2b molecular species of high molecular weight was detected, even after immunoaffinity chromatography and characterized by mass spectrometry and automatic sequencing. This HuIFN alpha 2b species was synthesized by an inefficient reading of the UGA natural stop codon, stopping the translation at another UGA in frame placed 10 codons downstream of the HuIFN alpha 2b stop signal. To avoid this translational readthrough process the UGA termination codon was replaced by UAA, which is frequently used in highly expressed E. coli genes. Simultaneously, almost all the HuIFN alpha 2b gene 3' noncoding region was removed. Analysis by SDS-PAGE and enzyme-linked immunosorbent assay revealed the elimination of the undesired HuIFN alpha 2b molecular species and an almost twofold increase in the expression level. These results indicate that both factors, the stop codon used and the length of the transcription unit should be taken into account when the expression in E. coli of heterologous proteins is desired.
César Sánchez
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Center for Genetic Engineering and Biotechnology, La Habana, Cuba. Santana@cigb.edu.cu
Padrón
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Santana
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Herrera
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D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D018388:Codon, Terminator / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010443:Peptide Chain Termination, Translational / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011994:Recombinant Proteins
D018388:Codon, Terminator; D017931:DNA Primers; D004274:DNA, Recombinant; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins