Patent ID: 8771984
Filing Date: 2014-07-08
Classification: C07K

Abstract:
1. A method for utilizing a multicistronic transcription unit to produce a peptide of interest from a host cell, the method comprising: introducing into a plurality of host cells of a eukaryotic cell line a DNA molecule comprising, in 5′-3′ direction: a promoter, a bicistronic transcription unit comprising: a first upstream polynucleotide with the translation start codon selected from the group consisting of GTG and TTG, wherein the first upstream polynucleotide encodes a selectable marker polypeptide with no methionine amino acids in its sequence that provides resistance against the lethal or growth-inhibitory effects of a selection marker, and a second downstream polynucleotide encoding the peptide of interest, wherein the second downstream polynucleotide has the translation initiation sequence ATG,  a transcription termination sequence,  wherein the DNA molecule further comprises at least two anti-repressor (STAR) sequences flanking the bicistronic transcription unit, wherein the at least two anti-repressor (STAR) sequences flanking the bicistronic transcription unit are a STAR7 sequence (SEQ ID NO:7) and a STAR67 sequence (SEQ ID NO:66) located upstream of the promoter, and a STAR7 sequence (SEQ ID NO:7) located downstream of the transcription termination sequence; culturing the plurality of host cells to select for expression of the selectable marker polypeptide; selecting at least one host cell producing the peptide of interest; and producing the peptide of interest from the host cell.