Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms

Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the moss Physcomitrium patens generates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encoding Physcomitrella genes and their 87,533 annotated transcripts in a web application, physCO, for automatized optimization. A thus optimized cDNA results in about twelve times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects occur also in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology. Top et al. report the expression of a human blood-clotting factor IX-encoding cDNA in the moss Physcomitrium patens and demonstrate that in addition to the unspliced full-length transcript, the splicing machinery produced several different transcripts, and thus different protein isoforms, which the authors designate as heterosplicing. The authors present a web application, physCO, to automatize the process of P. patens-specific codon optimization, a tool that can assist in maximized functional protein amounts for biotechnology.

Automated summary

The expression of human cDNA in moss generates unexpected transcripts, negatively impacting protein expression. By analyzing genes and transcripts, a web application called physCO was developed to optimize cDNA for increased protein yield. This research can assist in maximizing functional protein amounts in biotechnology.