Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.     

Nucleocytoplasmic shuttling of antigen in mammalian cells conferred by a soluble versus insoluble single-chain antibody fragment equipped with import/export signals

The ectopic expression of antibody fragments within mammalian cells is a challenging approach for interfering with or even blocking the biological function of the intracellular target. For this purpose, single-chain Fv (scFv) fragments are generally preferred. Here, by transfecting several mammalian cell lines, we compared the intracellular behavior of two scFvs (13R4 and 1F4) that strongly differ in their requirement of disulphide bonding for the formation of active molecules in bacteria. The scFv 13R4, which is correctly folded in the bacterial cytoplasm, was solubly expressed in all cell lines tested and was distributed in their cytoplasm and nucleus, as well. In addition, by appending to the 13R4 molecules the SV40 T-antigen nuclear localisation signal (NLS) tag, cytoplasmic-coexpressed antigen was efficiently retargeted to the nucleus. Compared to the scFv 13R4, the scFv 1F4, which needs to be secreted in bacteria for activity, accumulated, even with the NLS tag, as insoluble aggregates within the cytoplasm of the transfected cells, thereby severely disturbing fundamental functions of cell physiology. Furthermore, by replacing the NLS tag with a leucine-rich nuclear export signal (NES), the scFv 13R4 was exclusively located in the cytoplasm, whereas the similarly modified scFv 1F4 still promoted cell death. Coexpression of NES-tagged 13R4 fragments with nuclear antigen promoted its efficient retargeting to the cytoplasm. This dominant effect of the NES tag was also observed after exchange of the nuclear signals between the scFv 13R4 and its antigen. Taken together, the results indicate that scFvs that are active in the cytoplasm of bacteria may behave similarly in mammalian cells and that the requirement of their conserved disulphide bridges for activity is a limiting factor for mediating the nuclear import/export of target in a mammalian cell context. The described shuttling effect of antigen conferred by a soluble scFv may represent the basis of a reliable in vivo assay of effective protein- protein interactions.     

Growth signalobody selects functional intrabodies in the mammalian cytoplasm

A versatile strategy to inhibit protein functions in the cytoplasmic environment is eagerly anticipated for drug discovery. In this study, we demonstrate a novel system to directly select functional intrabodies from a library in the mammalian cytoplasm. In this system, a target homo‐oligomeric antigen is expressed together with a single‐chain Fv (scFv) library that is linked to the cytoplasmic domain of a receptor tyrosine kinase (RTK) in the cytoplasm of murine interleukin‐3 (IL‐3)‐dependent cells. As the tyrosine kinase is activated by dimerization, only scFv‐RTK clones that can bind to the target antigen would be oligomerized and transduce a growth signal under the IL‐3‐deprived condition, which leads to selection of functional intrabodies. To demonstrate this system, we used rabies virus phosphoprotein (RV‐P) that forms dimers in the cytoplasm as a target antigen. As a result, functional intrabodies were selected using our system from a naïve scFv library as well as from a pre‐selected anti‐RV‐P library generated by phage display. This system may be applied for screening intrabodies that can prevent progression of various severe diseases.     

Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single‐chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti‐PCNA scFvs were screened in vitro after being tagged with dimeric glutathione‐S‐transferase. Anti‐PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error‐prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C‐terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide‐conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody‐based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright © 2014 John Wiley & Sons, Ltd.     

Engineering stable cytoplasmic intrabodies with designed specificity

Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the periplasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K(d)=15nM) to that of the cognate scFv(D1.3) fragment (K(d)=16nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm.     

Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)(n) repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.     

Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules

The use of antibodies in medicine and research depends on their specificity and affinity in the recogniton and binding of individual molecules. However, these applications are limited to the extracellular targets. Advances in antibody engineering has allowed the manipulation of the antibody segments containing the antigen-binding regions and generation of small fragments that can be stably expressed in cells. These entities are called intracellular antibodies or intrabodies and have being successfully applied, mainly in the scFv format, to inhibit the function of intracellular target proteins in specific cellular compartments. As new techniques to select and isolate intrabody fragments have been developed, intrabodies are beginning to be used to interfere with the function of a greater number of relevant disease targets. Just as monoclonal antibodies are opening a new era in human therapeutics, intrabodies promise a new prospective for antibody tools for therapy and research. Their varied mode of action gives intrabodies great potential in different approaches in the treatment of human diseases, as well as in the area of functional genomics for characterisation of novel gene products and subsequent validation as potential drug targets. While techniques for identifying functional intrabodies have improved, there are still many significant problems to be overcome before intrabodies can actually be used in treatment of diseases such as cancer, AIDS or neuro-degenerative disorders.     

Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL-2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.     

Intracellular antibody capture technology: application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein

The expression of antibodies inside cells to ablate protein function has the potential for disease therapy and for target validation in functional genomics. However, due to inefficient expression or folding, only a few antibodies or antibody fragments, usually as single-chain Fv antibody fragments (scFv), bind their antigens in an intracellular environment. We have established a genetic-selection technology (intracellular antibody capture, IAC) to facilitate the isolation of functional intracellular scFv from a diverse repertoire. This approach comprises an in vitro library screen with scFv-expressing bacteriophage, employing bacterially expressed antigen, followed by a yeast in vivo antibody-antigen interaction screen of the sub-library of in vitro scFv antigen-binders. Accordingly, we have isolated panels of scFv that bind intracellularly to the BCR or the ABL parts of the BCR-ABL oncogenic protein. Sequence analysis of the intracellular antibody scFv panels revealed a sequence conservation indicating an intracellular antibody consensus for both VH and VL, which could form the basis for the de novo synthesis of intracellular antibody libraries to be used with intracellular antibody-capture technology.     

Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies

There is a major need in target validation and therapeutic applications for molecules that can interfere with protein function inside cells. Intracellular antibodies (intrabodies) can bind to specific targets in cells but isolation of intrabodies is currently difficult. Intrabodies are normally single chain Fv fragments comprising variable domains of the immunoglobulin heavy (VH) and light chains (VL). We now demonstrate that single VH domains have excellent intracellular properties of solubility, stability and expression within the cells of higher organisms and can exhibit specific antigen recognition in vivo. We have used this intracellular single variable domain (IDab) format, based on a previously characterised intrabody consensus scaffold, to generate diverse intrabody libraries for direct in vivo screening. IDabs were isolated using two distinct antigens and affinities of isolated IDabs ranged between 20 nM and 200 nM. Moreover, IDabs selected for binding to the RAS protein could inhibit RAS-dependent oncogenic transformation of NIH3T3 cells. The IDab format is therefore ideal for in vivo intrabody use. This approach to intrabodies obviates the need for phage antibody libraries, avoids the requirement for production of antigen in vitro and allows for direct selection of intrabodies in vivo.     

Production of fluorescent single-chain antibody fragments in Escherichia coli

We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.     

Identification of a short amino acid sequence essential for efficient nuclear targeting of the Epstein-Barr virus nuclear antigen 3A.

The Epstein-Barr virus nuclear antigen 3A is expressed in the nuclei of cells latently infected by the Epstein-Barr virus. We have previously shown that a fragment of 265 amino acids was essential for the proper subcellular localization of the Epstein-Barr virus nuclear antigen 3A. As described in this paper, we have used deletion analysis to identify a decapeptide, RDRRRNPASR, which is essential for nuclear localization of this protein. Furthermore, this decapeptide is a functional nuclear localization signal as demonstrated by its ability to target expression of beta-galactosidase in the nuclei of transfected cells.     

Paracrine inhibition of prion propagation by anti-PrP single-chain Fv miniantibodies

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C). A growing body of evidence suggests that antibodies to PrP(C) can antagonize deposition of PrP(Sc). However, host tolerance hampers the induction of immune responses to PrP(C), and cross-linking of PrP(C) by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP(Sc), even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases.     

Intracellular stability of anti-caspase-3 intrabodies determines efficacy in retargeting the antigen.

Although intracellular antibodies (intrabodies) are being explored as putative therapeutic and research reagents, little is known about the principles that dictate the efficacy of these molecules. In our efforts to address this issue, we generated a panel of five intrabodies, directed against catalytically inactive murine caspase-3, by screening single-chain antibody (Fv) phage display libraries. Here we determined criteria that single-chain Fv fragments must fulfill to act as efficient intrabodies. The affinities of these intrabodies, as measured by surface plasmon resonance, varied approximately 5-fold (50-250 nm). Despite their substantial sequence similarity, only two of the five intrabodies were able to significantly accumulate intracellularly. These disparities in intracellular expression levels were reflected by differences in the stability of the purified protein species when analyzed by urea denaturation studies. We observed varied efficiencies in retargeting the antigen murine caspase-3, from the cytosol to the nucleus, mediated by intrabodies tagged with an SV40 nuclear localization signal. Our results demonstrate that the intrinsic stability of the intrabody, rather than its affinity for the antigen, dictates its intracellular efficacy.     

Degradation of a nuclear-localized protein in mammalian COS cells, using Escherichia coli beta-galactosidase as a model protein.

To investigate the mechanism of degradation of proteins localized in the nucleus, we constructed genes encoding modified Escherichia coli beta-galactosidases and expressed them in mammalian COS cells. When the beta-galactosidase with a nuclear localization signal from SV 40 T antigen was expressed in COS cells, the beta-galactosidase polypeptide was localized in the nuclei and was stable for at least 4 h. When 16 amino acid residues were deleted from the C-terminal end, the beta-galactosidase polypeptide was also observed in the nuclei but it was degraded rapidly, with a half-life of 1.6 h. When the nuclear localizing signal was replaced with a mutant sequence, which lacks nuclear targeting activity, the beta-galactosidase polypeptides were present throughout the cells rather than in the nuclei. The beta-galactosidase polypeptide with the complete C terminus was stable and the cytoplasmic truncated polypeptide was degraded at the same rate as the nuclear C terminus truncated polypeptide. The beta-galactosidase polypeptides with the complete C terminus were present as a tetramer as reported previously and had beta-galactosidase activity, but the C terminus truncated polypeptides were present as monomer and had no enzyme activity, indicating that C terminus truncated beta-galactosidase is malfolded. Together, the results suggest that a nuclear-localized malfolded protein is degraded as rapidly as a cytoplasmic malfolded protein.     

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.     

Generation of functional scFv intrabodies for triggering anti-tumor immunity

Intracellular expression of recombinant antibodies (intrabodies) allows to interfere with the functions of oncogenic or viral molecules expressed in different cell compartments and has therefore a vast clinical potential in therapy. Although the use of phage-display libraries has made it possible to select Fab or single chain Fv (scFv) antibody fragments usable for intracellular targeting, a major source of recombinant antibodies for therapeutic use still remains hybridoma B cells producing well-characterized monoclonal antibodies (mAbs). However, the cloning and the intracellular expression of antibody fragments derived from mAbs can be markedly hampered by a number of technical difficulties that include failure of cloning functional variable regions as well as lack of binding of the antibody fragments to the targeted molecule in an intracellular environment. We discuss herein various molecular methods that have been developed to generate functional recombinant antibody fragments usable as anti-tumor triggering agents when expressed in tumor cells. Such antibodies can neutralize or modify the activity of oncogenic molecules when addressed in specific subcellular compartments and/or they can be used to trigger anti-tumor immunity when expressed on tumor cell surface.     

A kinetic trap is an intrinsic feature in the folding pathway of single-chain Fv fragments

We have studied the equilibrium unfolding and the kinetics of folding and unfolding of an antibody scFv fragment devoid of cis-prolines. An anti-GCN4 scFv fragment carrying a VL lambda domain, obtained by ribosome display, served as the model system together with an engineered destabilized mutant in VH carrying the R66K exchange. Kinetic and equilibrium unfolding experiments indicate that the VH mutation also affects VL unfolding, possibly by partially destabilizing the interface provided by VH, even though the mutation is distant from the interface. Upon folding of the scFv fragment, a kinetic trap is populated whose escape rate is much faster with the more stable VH domain. The formation of the trap can be avoided if refolding is carried out stepwise, with VH folding first. These results show that antibody scFv fragments do not fold by the much faster independent domain folding, but instead form a kinetically trapped off-pathway intermediate, which slows down folding under native conditions. This intermediate is characterized by premature interaction of the unfolded domains, and particularly involving unfolded VH, independent of proline cis-trans isomerization in VL. This work also implies that VH should be a prime target in engineering well behaving antibody fragments.     

Specific in vivo knockdown of protein function by intrabodies

Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed.     

In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner

Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.