Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichi coli
2007 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 5Article in journal (Refereed) Published
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 SID-like sequences. Possible mechanisms behind the observed effects are discussed.
Place, publisher, year, edition, pages
2007. Vol. 35, no 5
RIBOSOME-BINDING-SITE; POLYMERASE-CHAIN-REACTION; HIGH-LEVEL EXPRESSION; TRANSLATION INITIATION; MESSENGER-RNA; CODON USAGE; SILENT MUTATIONS; HETEROLOGOUS PROTEINS; SECONDARY STRUCTURE; GENE-EXPRESSION
IdentifiersURN: urn:nbn:se:kth:diva-6924DOI: 10.1093/nar/gkl1171ISI: 000246371200035ScopusID: 2-s2.0-34247162596OAI: oai:DiVA.org:kth-6924DiVA: diva2:11774
QC 201006232007-03-272007-03-272012-03-19Bibliographically approved