Utilization of non‐AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines

Abstract

Here we describe a method that couples flow cytometric detection with the attenuated translation of a reporter protein to enable efficient selection of CHO clones producing high levels of recombinant proteins. In this system, a small cell surface reporter protein is expressed from an upstream open reading frame utilizing a non-AUG initiation (alternate start) codon. Due to the low translation initiation efficiency of this alternate start codon, the majority of translation initiation events occur at the first AUG of the downstream open reading frame encoding the recombinant protein of interest. While translation of the reporter is significantly reduced, the levels are sufficient for detection using flow cytometric methods and, in turn, predictive of protein expression from the gene of interest since both ORFs are translated from the same mRNA. Using this system, CHO cells have been sorted to obtain enriched pools producing significantly higher levels of recombinant proteins than the starting cell population and clones with significantly better productivity than those generated from limiting dilution cloning. This method also serves as an effective screening tool during clone expansion to enable resources to be focused solely on clones with both high and stable expression.

Generation of monoclonal antibody-producing mammalian cell lines

“…ClonePix™ from Genetix [202,203] and CellCelector from Aviso [204] are two systems that have been used for selecting recombinant protein-producing colonies. Cells expressing mAb are plated in semi-solid media containing a fluorescent detection antibody against the…”

uORFdb—a comprehensive literature database on eukaryotic uORF biology

“…Nevertheless, uAUG codons appeared to be functionally most effective in repressing CDS translation. To date, only two publications analyzing human BIRC2 and yeast GCN4 have been focusing on non-AUG uORF functions at the individual transcript level (23,24). …”

Fission yeast APC/C activators Slp1 and Fzr1 sequentially trigger two consecutive nuclear divisions during meiosis

“…Therefore, it is likely that Slp1 was indeed translated from the ATA codon at reduced levels, and that the reduced amounts of Slp1 are sufficient for the vegetative cell cycle but insufficient for meiosis. Although non‐ATG initiation of translation has been reported in other organisms , this is the first such example in S. pombe .…”