CloneOpt clones reduce production costs and make downstream purification steps easier

A beginners guide to translation initiation

Translation initiation is the first step of protein synthesis. Here the 30S subunit of the ribosome recognises a region of the mRNA known as the translation initiation region (TIR) and completes the assembly of a fully functional ribosome. 

The sequence of the TIR directly influences the efficiency of translation initiation. It typically contains a Shine-Dalgarno sequence upstream of the start codon, which base pairs with the ribosome (Figure 1). Although there are in principle 15 billion sequence permutations in the TIR, nature has selected only a few thousand of these. Those that were selected have co-evolved with the ribosome and are therefore highly compatible with it. 

Figure 1. The TIR (green box) includes the SD sequence, the AUG start codon and the 5' end of the coding sequence. The sequence of the TIR directly influences the efficiency of translation initiation. Adapted from Reeve et al, Frontiers Bioeng. Biotech. 2014

When expression clones are produced in a laboratory settings the TIR is randomly synthesized from the expression vector (which harbours a Shine Dalgarno sequence) and the 5’ end of coding sequence. These two modules have not been under selection pressure, and are rarely optimal at recruiting host cell ribosomes.  As a consequence they do not function efficiently in initiating translation. 

CloneOpt technologies

CloneOpt uses proprietary technologies to ‘synthetically evolve’ a TIR so that it is highly compatible with host cell ribosomes. The process involves the generation of clone libraries that contain circa 50,000 TIRs. Protein production levels from each TIR are then experimentally evaluated and the most efficient TIR is selected (Figure 2). 


Figure 2. CloneOpt synthetically evolves TIRs on expression clones


CloneOpt TIRs are guaranteed to produce higher production yields of recombinant proteins since they have been experimentally identified. Examples of how CloneOpt TIRS improve protein expression are presented in Figure 3. 


Figure 3. A membrane transport protein, expressed in BL21(DE3) from a pET28a vector.