Protein Production

About

Background: Stanford researchers have developed a novel method to tightly control recombinant protein production levels. This technology provides the genetic tools and method to engineer, specify, and evaluate a broad range of gene expression levels by controlling protein translation initiation in eukaryotes. Additionally, two genes of interest can be expressed independently at different specified levels from the same transcript. The conservation of translational machinery across eukaryotes allows this technology to be employed in any eukaryotic system, including mammals, insects, plants and yeasts. This technology enhances the optimization of recombinant cells used to produce valuable chemicals, including biofuels and therapeutic proteins and may eventually be adapted for gene therapy.   Stage of Research:  The research is on-going in the Wang lab and has been used to control expression of fluorescent proteins, p21, Ras, and p53. Figures: Precise control of protein expression levels. (A) The translation level of a protein-coding open reading frame (ORF) is specified by varying bases comprising the translation initiation site (TIS) of upstream open reading frames (uORF) (NNNu) and TIS bases of the protein-coding ORF (NNNp) . Translation level can be further tuned by employing multiple uORFs in series (in parentheses with subscript n). 5’m, 5’-methylated mRNA cap; AAA, mRNA poly-A tail. Arrows indicates paths of ribosomes. (B) RNA leader sequences employing different uORFs and TISs mediated a range of GFP translation levels in HCT-116 cells. Leaders with varying TIS sequences and no uORFs were used to generate higher expression levels. Leaders utilizing different TIS sequences and uORFs were used to generate lower expression levels. Reporter translation was reported as GFP normalized to an internal RFP reference. (C) RNA leader sequences reproducibly generated a broad range of GFP expression levels across different cell types. Error bars represent standard deviations from triplicate experiments. (D) Immunoblot of a range of DHFR-BFP-RasG12V achieved using engineered RNA leaders. Expression was achieved in NIH-3T3 cells with (+) and without (-) trimethoprim, the small molecule inducer of DHFR fusion protein stability. For panels B and D, sequences of the RNA leaders are given with uORFs in red bold type (including stop codons); start codons are underlined; the first base of the start codon encoding a protein of interest is indicated by the +1 base position   Applications: Molecular and cellular biology: Study of molecular dose effects. Evaluation fo genes at physiologically relevant levels. Study of molecular dose effects. Evaluation of genes at physiologically relevant levels. Engineering: tuning or optimization of genes involved in: signaling pathways, metabolic pathways, transcription factors (stem cell differentiation) and synthetic genetic circuits. Impact on the fields of: basic research; cells for industrial applications (e.g., production, drug screening); programming of cell behavior and fate (i.e., stem cell differentiation); gene therapy.   Advantages: User friendly: simple and easy to implement. Broad range of control: up to 3 orders of magnitude expression. No competing technology enables precise specification of low-end expression levels. Broad platform technology with reproducible expression across cell types; species; yeast, plants, insect cells (with likely minimal or even no modification to technology) 200-600 fold range of expression in protein production compared to 20-40 fold by current methods Allows independent control of two different genes from the same mRNA transcript allowing the gene of interest to be expressed at a level independent of selection gene or cellular marker Reproducible expression across cell types eliminating the need for re-engineering for different cell types