Date Published: April 23, 2019
Publisher: Public Library of Science
Author(s): Jennifer Konczal, Justin Bower, Christopher H. Gray, Paulo Lee Ho.
Gene synthesis services have largely superseded traditional PCR methods for the generation of cDNAs destined for bacterial expression vectors. This, in turn, has increased the application of codon-optimized cDNAs where codons rarely used by Escherchia coli are replaced with common synonymous codons to accelerate translation of the target. A markedly accelerated rate of expression often results in a significant uplift in the levels of target protein but a substantial proportion of the enhanced yield can partition to the insoluble fraction rendering a significant portion of the gains unavailable for native purification. We have assessed several expression attenuation strategies for their utility in the manipulation of the soluble fraction towards higher levels of soluble target recovery from codon optimized systems. Using a set of human small GTPases as a case study, we compare the degeneration of the T7 promoter sequence, the use of alternative translational start codons and the manipulation of synonymous codon usage. Degeneration of both the T7 promoter and the translational start codon merely depressed overall expression and did not increase the percentage of product recovered in native purification of the soluble fraction. However, the selective introduction of rare non-optimal codons back into the codon-optimized sequence resulted in significantly elevated recovery of soluble targets. We propose that slowing the rate of extension during translation using a small number of rare codons allows more time for the co-translational folding of the nascent polypeptide. This increases the proportion of the target recovered in the soluble fraction by immobilized metal affinity chromatography (IMAC). Thus, a “de-optimization” of codon-optimized cDNAs, to attenuate or pause the translation process, may prove a useful strategy for improved recombinant protein production.
Widely available and affordable gene synthesis services have resulted in a decline in the use of PCR for the generation of recombinant expression clone inserts . Along with this comes a ready opportunity to manipulate the nucleotide sequence of these open reading frames, removing codons that correspond to rare tRNAs in the expression host that may prove a bottleneck to protein expression . The application of codon-optimized cDNAs is now prominent and often results in substantially improved output in protein production , and this trend is observed in our own E.coli expression laboratory. However, we consistently notice that a substantial proportion of the uplift achieved by codon-optimized cDNAs then partitions into the insoluble fraction, rendering it of poor quality and unavailable for native purification . Troubleshooting this solubility issue using the classic optimisation variables (media type, E.coli strain, concentration of inducing reagent, temperature etc. [5, 6]) had no substantial effect in these systems. This led us to formulate a hypothesis that the markedly accelerated expression in codon-optimized systems somehow overwhelms the kinetics of protein folding meaning that much of the additional target generated partitions to inclusion bodies.
The use of codon-optimized reading frames aims to eliminate any translational bottleneck imposed by a shortage of rare tRNAs as a consequence of the differing codon usage profile between the originating species of the target protein and that of the expression host cell . As a result, the synthetic mRNA transcript is scanned and processed by the ribosome very efficiently, maximising the extension rate of the nascent polypeptide . However, this accelerated expression rate can be too potent, resulting in a substantial amount of the increased yield partitioning to the insoluble fraction. Often, the translation rate of these optimized transcripts overwhelms the co-translational folding kinetics of the target protein . As a result, the increased gain from codon-optimized cDNAs is somewhat frustrated.
We had previously noted that the expression kinetics from our codon-optimized vectors often seemed “too hot”, pushing the gains into the insoluble fraction. However, conventional optimisation methods did not assist. These data suggest that the reason for the solubility issues did not reside in over production of transcript or an over-frequent engagement with the ribosome to initiate translation. Rather, the fully optimized transcript was scanned by the ribosome too efficiently, unfettered by any restriction in the tRNA population. As a consequence the extension rate of the nascent polypeptide outpaced the kinetics of co-translational folding resulting in a frustrating increase in insoluble material. By re-introducing a small number of rare codons back into the optimised cDNA, the translation rate is somewhat slowed, allowing more productive co-translational folding and substantially increasing soluble yields recovered by native IMAC purifications.