Anchored periplasmic expression (APEx): a versatile technology for the flow cytometric selection of high affinity antibodies from Escherichia coli expressed libraries



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Recombinant proteins are increasingly being used in health care over a broad spectrum of applications, ranging from cancer treatment to microbial infections. Antibodies in particular are showing promise as therapeutics and diagnostic tools. To keep up with the demand for antibodies with tailored activity, evolutionary methods have been utilized that allow for the discovery of protein function without the need for an accurate understanding of structure to function relationship as required by rational design. This dissertation describes the development and implementation of a new combinatorial protein library screening strategy, referred to as APEx (for Anchored Periplasmic Expression). APEx is based on anchoring proteins, such as single chain variable fragment antibodies (scFv), to the periplasmic face of the inner membrane of Escherichia coli using novel fusion strategies. For example, the first six amino acids of the mature lipoprotein, new lipoprotein A (NlpA), which is fatty acylated and anchored to the outside of the inner membrane upon export, proved to be an ideal display partner. Upon chemical permealization of the outer membrane, cells expressing NlpA(1-6)-scFv protein fusions can be readily labeled with fluorescently conjugated antigens that include small molecules, peptides and proteins that can be as large as 240 kDa in size. This allows for scFv antibody libraries displayed by APEx to be screened by flow cytometry. Antibodies to a bacterial toxin subunit, namely the Protective Antigen protein (PA) component of the Bacillus anthracis toxin, and drugs of abuse such as methamphetamine derivatives, have been engineered for affinity improvement. Notably, the affinity of an scFv antibody specific for PA was enhanced 120 fold to 35pM, following one round of mutagenesis of the parental antibody and screening of the resulting library by APEx. In addition to affinity improvement, isolated antibodies exhibit excellent expression characteristics, likely because bacterial expression is an implicit criterion in the selection process and the short six amino acid residue extension used for APEx does not impact the expression characteristics of the fused target protein.