Nucleic Acids Research Advance Access originally published online on January 30, 2007
Nucleic Acids Research 2007 35(5):e32; doi:10.1093/nar/gkl1171
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Nucleic Acids Research, 2007, Vol. 35, No. 5 e32
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichia coli
Department of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden
*To whom correspondence should be addressed. Tel: +46 8 55378328; Fax: +46 8 55378481; Email: perake{at}biotech.kth.se
Received November 6, 2006. Revised December 21, 2006. Accepted December 21, 2006.
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.
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