Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichia coli.
The complex and integrated nature of both genetic and protein level factors influencing recombinant protein production in Escherichia coli makes it difficult to predict the optimal expression strategy for a given protein. Here, two combinatorial library strategies were evaluated for their capability of tuning recombinant protein production in the cytoplasm of E. coli. Large expression vector libraries were constructed through either conservative (ExLib1) or free (ExLib2) randomization of a seven-amino-acid window strategically located between a degenerated start codon and a sequence encoding a fluorescently tagged target protein. Flow cytometric sorting and analyses of libraries, subpopulations or individual clones were followed by SDS-PAGE, western blotting, mass spectrometry and DNA sequencing analyses. For ExLib1, intracellular accumulation of soluble protein was shown to be affected by codon specific effects at some positions of the common N-terminal extension. Interestingly, for ExLib2 where the same sequence window was randomized via seven consecutive NN(G/T) tri-nucleotide repeats, high product levels (up to 24-fold higher than a reference clone) were associated with a preferential appearance of novel SD-like sequences. Possible mechanisms behind the observed effects are discussed.
Bandmann
Nina
N
Department of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
Nygren
Per-Ake
PA
D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D015870:Gene Expression; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D010442:Peptide Chain Initiation, Translational; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D003062:Codon; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins