Enhancing translational efficiency for in vitro protein expression
Biomax teamed up with an industrial partner, Roche Diagnostics GmbH, to investigate and improve the efficiency of translation with the RTS in vitro expression system. The system, which provides a number of advantages over cell-based systems, is designed to maximize yields in high-throughput protein expression systems.
A polymerase chain reaction (PCR) based method was established to generate hundreds of different expression cassettes for quantitative evaluation in a coupled E. coli in vitro transcription translation system. The influence of sequence variations in the translated region of the mRNA were examined in a large-scale expression experiment involving 756 expression constructs. Statistical analysis identified sequence regions that have a profound effect on translational efficiency and indicated that mRNA secondary structure in these regions decreases efficiency. Training data were used to develop a method to predict and improve the translation efficiency of open reading frames (read more).
Using a secure data connection, the user transfers the sequence to be translated to Biomax. The sequence alterations are calculated to identify changes that exhibit a high probability of improving expression yield without changing the amino acid sequence of the translation product. Furthermore, the user is provided with all needed primer sequences and detailed descriptions for generating the expression construct in the laboratory. The generated linear constructs can be used directly for rationally optimized in vitro expression experiments.
Selected sequence parameters (ppav and gcav) are plotted against observed expression levels for tested constructs to derive scores for predicting expression levels for untested sequences.
The following Biomax solutions were used in preparing and performing the analysis: