2G UNic™

Recombinant protein production in cultured cells
Cultured cells can be reprogrammed to produce specific proteins of commercial interest by insertion of new genes into the cell genome. Following this process, the gene is transcribed into RNA (transcription), which in turn is used as a template for protein production (translation). The newly synthesized protein molecules are transported through the cell and – in most cases – secreted into the culture medium from which they can be isolated. The various steps in this process impact overall recombinant protein production levels, and need to be optimized for efficient, high level production. Methods for increased transcription are well established and broadly used. Until recently, it was not generally recognized that translation can be optimized to boost recombinant protein production.


2G UNic™
ProteoNic has identified DNA elements which are instrumental in increasing translation rates when included in recombinant protein expression vectors. These Translation Enhancing Elements (TEE-s) are functional across eukaryotic production platforms, including mammalian cells, yeast, and most fungal systems. Based on these TEE-s, ProteoNic has developed its proprietary 2G UNic™ technology.

Mechanism of action of 2G UNic™
The start of translation is the rate limiting factor in the translation cycle. The key step in translation initiation is the assembly of a ribosome (the core of the protein synthesizing machinery) on the mRNA molecule. The efficiency of ribosome assembly is mainly determined by the mRNA sequence at and around the assembly site, upstream of the coding sequence of the gene to be expressed. By the functional introduction of a TEE at the appropriate site, the affinity of ribosomes for this region can be increased significantly. Via in vitro translation studies based on the use of cell extracts it has been shown that ribosome binding affinity can be increased 50-fold by incorporation of TEE-s. This increased affinity results in a higher number of ribosomes binding and starting translation for a given protein. The overall effect is an increase in translation rate and  more protein production per TEE containing mRNA template.

Improved translation efficiency contributes to increased recombinant protein production.
Using a range of mammalian, yeast and fungal cell types, the functionality of 2G UNic™ technology has been confirmed for a variety of industry relevant platforms. Depending on initial expression levels, expression of secreted recombinant proteins can be increased several-fold, and is coupled to a similar increase in recombinant protein to mRNA ratios.
An example for CHO cells is shown in figure 2. Using site directed integration to rule out integration site effects, single copy expression levels of human alkaline phosphatase (SeAP) increase 2.5-fold due to TEE incorporation and subsequent improved protein to mRNA ratios.


Functional incorporation of 2G UNic™ into expression vectors leads to increased translation rates due to improved ribosome recruitment. As a result, more recombinant protein is produced per amount of mRNA.