title: Enhanced yield of recombinant proteins with site-specifically incorporated unnatural amino acids using a cell-free expression system.
abstract: Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 µg/mL of translated protein (comprising 25 µg in 50 µL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.
authors.affiliation: Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Sviatlana
authors.initials: S
authors.lastname: Smolskaya
authors.forename: Zhiwen Jonathan
authors.initials: ZJ
authors.lastname: Zhang
authors.forename: Lital
authors.initials: L
authors.lastname: Alfonta
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002474:Cell-Free System / Q000378:metabolism*; D018388:Codon, Terminator / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D018388:Codon, Terminator; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine