PEDS Advance Access published online on May 3, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzl016
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1 Department of Protein Therapeutics, Diversa Corp. 4955 Directors Place, San Diego, CA 92121, USA; Present address: Biogen Idec, 5200 Research Place, San Diego, CA 92122, USA
* To whom correspondence should be addressed. The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92°C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.
Received August 15, 2005
Revised January 1, 2006
Accepted March 27, 2006
Article
Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression
Stephen J. Demarest 1 *,
Gang Chen 2,
Bruce E. Kimmel 3,
David Gustafson 2,
Jane Wu 2,
Jared Salbato 2,
John Poland 2,
Marikka Elia 2,
Xuqiu Tan 2,
Ken Wong 2,
Jay Short 4,
and
Geneviève Hansen 5
2 Department of Protein Therapeutics, Diversa Corp. 4955 Directors Place, San Diego, CA 92121, USA
3 Department of Protein Therapeutics, Diversa Corp. 4955 Directors Place, San Diego, CA 92121, USA; Present address: Ambrx, Inc., 10410 Science Center Drive, San Diego, CA 92121, USA
4 Department of Protein Therapeutics, Diversa Corp. 4955 Directors Place, San Diego, CA 92121, USA; Present address: E.O. Wilson Biodiversity Foundation, 10190 Telesis Court, San Diego, CA 92121, USA
5 Department of Protein Therapeutics, Diversa Corp. 4955 Directors Place, San Diego, CA 92121, USA; Present address: Lpath Therapeutics, Inc., 5335 Ferris Sq., Suite A. San Diego, CA 92121, USA
Stephen J. Demarest, E-mail: stephen.demarest{at}biogenidec.com
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