Browsing by Subject "Recombinant proteins"
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Item Anchored periplasmic expression (APEx): a versatile technology for the flow cytometric selection of high affinity antibodies from Escherichia coli expressed libraries(2003) Harvey, Barrett Rowland; Georgiou, GeorgeRecombinant 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.Item Antibody discovery and engineering using the anchored periplasmic expression (APEx) Escherichia coli display system with flow cytometric selection(2009-08) Van Blarcom, Thomas John; Georgiou, GeorgeThe development of recombinant proteins for therapeutic applications has revolutionized the pharmaceutical industry. In particular, monoclonal antibodies are the safest class of all therapeutic molecules and account for the majority of recombinant proteins currently undergoing clinical trials. A variety of technologies exist to engineer antibodies with a desired binding specificity and affinity, both of which are a prerequisite for therapeutic applications. This dissertation describes the implementation of a novel combinatorial library screening technology for the discovery and engineering of antibodies with unique binding properties. Combinatorial library screening technologies are used for the in vitro isolation of antibodies from large ensembles of proteins (libraries) typically produced by microorganisms using molecular biology techniques. Our lab has developed a powerful antibody discovery technology that relies on E. coli display by anchored periplasmic expression, otherwise known as APEx. First, I compared the effects of using combinatorial libraries comprising either smaller, monovalent single-chain antibody fragments (scFv), or the much larger, bifunctional full-length IgG antibodies. These technologies were used to isolate a small panel of antigen specific antibodies from the same library of antibody variable domains amplified from a mouse immunized with the Protective Antigen (PA) component from Bacillus anthracis, the causative agent of anthrax. Overall, IgG display resulted in the isolation of a broader panel of variable domain sequences. Most of these variable domains exhibited substantially reduced affinity when expressed as scFvs, which is consistent with the finding that none of these could be isolated from the equivalent scFv library. These results indicate that the antibody format used during in vitro selection affects which antibody variable domains will be discovered. Second, I developed several modifications of the APEx methodology to allow for more efficient recovery of antibodies with desired properties. Specifically, the system was reengineered to simultaneously account for antibody binding and expression levels in order to isolate the highest affinity antibodies with favorable expression characteristics. Third, the new approach, coupled with optimized fluorescence activated cell sorting (FACS) settings, was used to increase the affinity of an antibody by 35-fold resulting in a K[subscript D] of 100 pM. It was demonstrated that genetic transfer of this high affinity antibody specific for the V antigen of Yersinia pestis, the etiologic agent of the plague, conferred increased protection against intranasal challenge with a 363 LD₅₀ of Y. pestis in mice.