title: High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins.
abstract: We describe a high-throughput strategy for tuning the expression of recombinant proteins through engineering their early nucleotide sequences. After randomizing the +2 and +3 codons of the target genes, each of the variant genes was isolated in vivo and subsequently expressed using in vitro protein synthesis techniques. When several hundreds of clones were examined in parallel, it was found that expression levels of target genes varied as much as 70-fold depending on the identity of the codons in the randomized region. This broad and continuous distribution of expression levels enabled the selection of specific codon arrangements for the expression of target genes at a desired level. Furthermore, codon-dependent variations in protein expression were reproduced when the same genes were expressed in vivo. Thus, we expect that the methodology reported here could be utilized as a versatile platform for rapid expression of protein molecules at modulated levels either in vitro or in vivo.
authors.affiliation: Institute of Molecular Biology and Genetics, School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Korea.
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Ahn
authors.forename: Jung-Won
authors.initials: JW
authors.lastname: Keum
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D003062:Codon; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011897:Random Allocation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins