DNA sequencing of 10 clones selected at random revealed an average of 1
DNA sequencing of 10 clones selected at random revealed an average of 1.8% nucleotide substitutions Mouse monoclonal to S1 Tag. S1 Tag is an epitope Tag composed of a nineresidue peptide, NANNPDWDF, derived from the hepatitis B virus preS1 region. Epitope Tags consisting of short sequences recognized by wellcharacterizated antibodies have been widely used in the study of protein expression in various systems. per gene. proteolysis, and aggregation.2,4,5 The expression of antibody fragments within intracellular compartments (intrabodies) constitutes a promising and clinically relevant technology for binding to target proteins relevant to disease progression.5 Intrabodies are being investigated as a potential treatment for human viral infection, cancer therapy, and neurodegenerative diseases.5C8 However, the cytoplasm of eukaryotic and most prokaryotic cells is maintained in a highly reduced state that strongly disfavors the formation of disulfide bonds under physiological conditions. Consequently, most antibodies are not compatible with expression in that compartment and thus cannot be employed as intrabodies. Naturally occurring antibodies exhibiting high thermodynamic stability and antigen binding under conditions where disulfide CL 316243 disodium salt bonds cannot form are rare.9,10 Therefore, scFvs with desired antigen specificity and sufficiently high stability to be suitable for use as intrabodies need to be generated de novo. Intrabodies have been isolated CL 316243 disodium salt by screening repertoire libraries using high-throughput screening methods that interrogate antibody function under reducing conditions, such as yeast 2-hybrid, protein complementation assays, and ribosomal display.11C16 In contrast, filamentous phage display necessitates the secretion of antibodies into the oxidizing environment of the bacterial periplasmic space. Therefore, for intrabody applications, phage-derived antibodies to target antigens must be subjected to a second screen to isolate clones compatible with cytoplasmic expression.17 Alternatively, phage display has been employed successfully for the directed evolution of hyperstable antibody frameworks that in some cases can withstand expression in the reducing environment of the cytoplasm.1 In turn, natural or engineered hyperstable antibody frameworks have been used as scaffolds for the creation of large synthetic libraries containing randomized CDRs13,18C20 enabling the isolation of scFvs that are folded in the absence of disulfides. Alternatively, MBP-scFv fusions have been shown to exhibit significant activity when expressed in the cytoplasm of or mammalian cells.21 The bacterial periplasmic space is a highly oxidizing compartment that strongly favors the formation of protein disulfide bonds. Cysteine oxidation is catalyzed by the highly efficient protein thiol oxidase DsbA.22,23 Upon transferring its disulfide to a substrate protein, DsbA becomes reduced and has to be recycled by the action of the membrane enzyme DsbB, which then transfers the electrons to quinones. In strains deficient in (or strains are not compatible with phage display because they do not support filamentous phage assembly.25 Earlier, we developed a flow cytometric technique for the screening of antibody fragments, called protein technologies is that binding affinity is directly and quantitatively measured at the single cell level by FACS. 28 In this work, we used APEX to screen libraries of scFvs that are able to fold into their native conformation in the reducing periplasm of cells. As a model system, we isolated variants of the anti-protective antigen (PA) 14B7* scFv that, in contrast to the parental antibody, were able to fold under reducing conditions and could be expressed in fully active form in the bacterial cytoplasm. Thus, the methodology we present here should enable the rapid isolation of antibody fragment variants that can fold into their active conformation under reducing conditions and can be used for intrabody applications. Open in a separate window Figure 1 Isolation of active antibody fragments in mutants. (A) Schematic showing the screening strategy. Left panel: a correctly folded scFv anchored on the inner membrane of wild-type cells is able to bind antigen. A fluorescent antibody that recognizes an epitope tag on the antigen is used to detect the formation of antibody:antigen complex. Middle panel: in the mutant, the scFv is reduced and cannot bind antigen. Upon spheroplasting, the antigen diffuses away and hence the cell is not labeled by the fluorescent anti-epitope tag antibody. CL 316243 disodium salt Right panel: a mutant scFv that is capable of folding in the absence of disulfide bonds can bind antigen in cells. M, mean fluorescence intensity of the cell population. (B) CL 316243 disodium salt Fluorescence histograms of cells coexpressing the 14B7* scFv and PA domain IV proteins in either MC1000 (wild-type) or MCA (MC1000 anti-FLAG-FITC. M, mean fluorescent intensity. (C) The redox state of the 14B7* scFv in MC1000 or MCA cells following alkylation with AMS and separation by nonreducing SDS-PAGE. Results Assay development As a model antibody fragment for this study, we used the 14B7* scFv that binds the Protective Antigen (PA) component of the toxin.30 In particular, the 14B7* scFv was anchored onto the inner membrane by fusion to the leader peptide and the first six amino acids of the mature sequence (CDQSSS) of the lipoprotein NlpA. The 14B7* scFv recognizes a conformational epitope located within PA domain 4 (PA-D4), a 139 amino acid fragment comprising aa 596C735.31 PA-D4 fused to a promoter.28 MC1000 cells expressing the membrane anchored 14B7* scFv together with periplasmic PA-D4 were converted to spheroplasts by treatment with lysozyme and EDTA in.