ELIMINATION OF AN HUIFN-ALPHA-2B READTHROUGH SPECIES, PRODUCED IN ESCHERICHIA-COLI, BY REPLACING ITS NATURAL TRANSLATIONAL STOP SIGNAL

When human interferon-alpha 2b (HuIFNalpha 2b) was expressed intracell ularly in Escherichia coli as insoluble aggregates, a HuIFNalpha 2b mo lecular species of high molecular weight was detected, even after immu noaffinity chromatography and characterized by mass spectrometry and a utomatic sequencing. This HuIFNalpha 2b species was synthesized by an inefficient reading of the UGA natural stop codon, stopping the transl ation 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 use d in highly expressed E. coli genes. Simultaneously, almost all the Hu IFNalpha 2b gene 3' noncoding region was removed. Analysis by SDS-PAGE and enzyme-linked immunosorbent assay revealed the elimination of the undesired HuIFNalpha 2b molecular species and an almost twofold incre ase in the expression level. These results indicate that both factors, the stop codon used and the length of the transcription unit should b e taken into account when the expression in E. coli of heterologous pr oteins is desired. (C) 1998 Published by Elsevier Science B.V. All rig hts reserved.