Protein engineering of streptavidin for in vivo assembly of streptavidin beads

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.     

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.     

Engineering of Escherichia coli beta-galactosidase for solvent display of a functional scFv antibody fragment

Protein engineering allows the generation of hybrid polypeptides with functional domains from different origins and therefore exhibiting new biological properties. We have explored several permissive sites in Escherichia coli beta-galactosidase to generate functional hybrid enzymes displaying a mouse scFv antibody fragment. When this segment was placed at the amino-terminus of the enzyme, the whole fusion protein was stable, maintained its specific activity and interacted specifically with the target antigen, a main antigenic determinant of foot-and-mouth disease virus. In addition, the antigen-targeted enzyme was enzymatically active when bound to the antigen and therefore useful as a reagent in single-step immunoassays. These results prove the flexibility of E. coli beta-galactosidase as a carrier for large-sized functional domains with binding properties and prompt the further exploration of the biotechnological applicability of the scFv enzyme targeting principle for diagnosis or other biomedical applications involving antigen tagging.     

Single-chain Fv with Fc fragment of the human IgG1 tag: construction, Pichia pastoris expression and antigen binding characterization

We report two expression vectors in Pichia pastoris that direct the synthesis of recombinant single chain antibody variable region (scFv), derived from anti-Z-DNA monoclonal antibody Z22. The first vector codes for a scFv fused to the Ig binding domain of staphylococcal Protein A. The second vector codes for the scFv fused to the Fc fragment of the human IgG1. The fusion partner simplified the detection and purification of the secreted protein. These constructs yielded high level expression of an scFv with specific binding activity toward a Z form of DNA, with binding activity comparable to that of the scFv molecule produced in an Escherichia coli expression system and the original monoclonal antibody.     

Specific drug binding by purified lipid-reconstituted P-glycoprotein: dependence on the lipid composition.

We fused P-glycoprotein with beta-galactosidase at the C-terminus aiming to study the mechanism of drug binding of P-glycoprotein in reconstitution experiments. Expression of the fusion protein in NIH 3T3 cells conferred a multidrug-resistant phenotype, suggesting that beta-galactosidase fusion at the C-terminus does not affect the functions of P-glycoprotein. The fusion protein was partially purified by simple immunoprecipitation with anti-beta-galactosidase polyclonal antibody, and its [3H]azidopine binding property was investigated in the presence of various compositions of liposomes. The purified P-glycoprotein, after reconstitution into liposomes, was capable of binding [3H]azidopine. When the cholesterol content of liposomes was increased to a weight ratio of 20%, the specific binding activity of the partially purified fusion protein was stimulated, and when the cholesterol content was increased higher, the binding activity decreased. The binding was specifically decreased by competition with vinblastine. Stigmasterol was less effective, and ergosterol was the least effective in stimulating the specific binding.     

An Escherichia coli plasmid vector system for production of streptavidin fusion proteins: expression and bioselective adsorption of streptavidin-beta-galactosidase

Covalently immobilized biotin was used as a biospecific adsorbant to investigate the application of streptavidin as an affinity domain for simultaneous purification and immobilization of recombinant proteins. A streptavidin-beta-galactosidase fusion protein was constructed and tested as a model system. The gene for streptavidin from Streptomyces avidinii was modified by polymerase chain reaction to mutate the stop codon and to facilitate cloning into an Escherichia coli expression vector yielding a versatile plasmid with 37 unique restriction enzyme sites at the 3' end. E. coli beta-galactosidase was cloned in-frame to the streptavidin gene. Analysis of lysates of induced recombinant E. coli cells by SDS-PAGE and Western blots indicated that the 133.6-kDa fusion protein was expressed. Sulfosuccinimidyl-6-(biotinamido) hexanoate was covalently immobilized on 3-aminopropyl-controled-pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin-beta-galactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 betamol/min per milligram protein with o-nitrophenyl-beta-D-galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli beta-galactosidase. (c) 1994 John Wiley & Sons, Inc.     

Multifunctional inorganic-binding beads self-assembled inside engineered bacteria

Multifunctional shell-core nano/microbeads with a hydrophobic biopolymer core and a designed protein coat for selective binding of an inorganic substance and antibodies were self-assembled inside engineered bacteria. Hybrid genes were constructed to produce tailormade bead-coating proteins in the bacterium Escherichia coli. These fusion proteins contained a binding peptide for an inorganic material, the antibody binding ZZ domain, and a self-assembly promoting as well as biopolymer synthesizing enzyme. Production of these multidomain fusion proteins inside E. coli resulted in self-assembly of beads comprising a biopolyester core and displaying covalently bound binding sites for specific and selective binding of an inorganic substance and any antibody belonging to the immunoglobulin G class. Engineered beads were isolated and purified from the respective E. coli cells by standard cell disruption procedures. Bead morphology and the binding functionalities displayed at the bead surface were assessed by the enzyme-linked immunosorbent assay, transmission electron microscopy, elemental analysis, backscattering electron density, analytical density ultracentrifugation, and atomic force microscopy. These analyses showed that bacteria can be engineered to produce fusion proteins mediating self-assembly of spherical biopolymer beads with binding affinity to gold and/or silica and antibodies. Spherical structures of this type could conceivably serve as nano/microdevices for bioimaging in medical approaches where an antibody mediated targeted delivery of an inorganic contrast agent would be desired.     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

抗MRP3单链抗体-sTRAIL融合蛋白诱导U251多形性胶质母细胞瘤凋亡

采用重组PCR技术获得抗多药耐药相关蛋白3(multidrug resistance protein 3, MRP3)的单链抗体(scFv)与人源可溶性肿瘤坏死因子相关凋亡诱导配体(soluble TNF-related apoptosis inducing ligand, sTRAIL)的融合蛋白质的基因编码序列, 利用原核表达载体pMAL-c2,构建含麦芽糖结合蛋白(maltose binding protein, MBP)标签肽的antiMRP3(scFv)-sTRAIL融合蛋白, 经亲和层析柱纯化. 获得纯化的antiMRP3(scFv)-sTRAIL融合蛋白,用MRP3阳性U251多形性胶质母细胞瘤做增殖抑制实验、细胞凋亡诱导实验,结果均显示具有明显的活性, 而MBP无明显作用. 上述结果表明,成功表达了antiMRP3(scFv)-sTRAIL融合蛋白, 该融合蛋白具有诱导U251多形性胶质母细胞瘤细胞凋亡的活性, 为开发靶向性抗肿瘤药物奠定了基础.     

Immobilization of active lipase B from <Emphasis Type="Italic">Candida antarctica</Emphasis> on the surface of polyhydroxyalkanoate inclusions

Polyhydroxyalkanoate (PHA) beads, recombinantly produced in Escherichia coli, were functionalized to display lipase B from Candida antarctica as translational protein fusion. The respective beads were characterized in respect to protein content, functionality, long term storage capacity and re-usability. The direct fusion of the PHA synthase, PhaC, to lipase B yielded active PHA lipase beads capable of hydrolyzing glycerol tributyrate. Lipase B beads showed stable activity over several weeks and re-usability without loss of function.     

Green fluorescent antibodies: novel in vitro tools

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs.

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.     

Preparation and application of anti-idiotypic antibody against anti-gibberellin A4 antibody.

A monoclonal anti-idiotypic antibody was raised against anti-gibberellin A4 (GA4) antibody, which recognizes biologically active gibberellins such as GA1 and GA4 specifically. Amino acid sequences of variable regions of both anti-GA4 and anti-idiotypic antibodies were analyzed. By using the property of the anti-idiotypic antibody to compete with GA1/4 in binding to the anti-GA4 antibody, we successfully applied the anti-idiotypic antibody to ELISA as a tracer for measuring GA1/4. The single-chain Fv (scFv) gene of the anti-idiotypic antibody was constructed, and scFv expressed in E. coli showed binding activity to anti-GA4 antibody. These results suggest the possible application of anti-idiotypic antibody as a handy and stable source of an enzymatic tracer for ELISA by production of fusion protein of the scFv and an appropriate enzyme.     

Beta-galactosidase: isolation of and antibodies to incomplete chains

A prematurely terminated polypeptide chain was purified to homogeneity from an Escherichia coli amber mutant strain containing the site of the mutation in the beta-galactosidase structural gene. The polypeptide was highly active against anti-beta-galactosidase, and had an amino acid composition similar to but not identical to that of beta-galactosidase. The molecular weight of the reduced, carboxymethylated chain in 6 m guanidine hydrochloride was found to be 89,000, in excellent agreement with the size predicted from the position of the mutation. This result adds further support to the conclusion that the gene specifies the structure of a single polypeptide chain. Antisera were prepared against partially purified preparations of this polypeptide and a similar one, of molecular weight about 100,000, produced by another amber mutant. These sera had lower titers towards beta-galactosidase than anti-beta-galactosidase. In the double-diffusion test, they reacted towards extracts of nonsense and deletion mutant strains in a pattern similar to that previously observed with anti-beta-galactosidase. A sensitive immunological test for cross-reacting protein was devised based on the inhibition by beta-galactosidase of the reaction between such protein and antibodies prepared against incomplete chains.     

A new model for intermediate molecular weight recombinant bispecific and trispecific antibodies by efficient heterodimerization of single chain variable domains through fusion to a Fab-chain

Due to their specificity and versatility in use, bispecific antibodies (BsAbs) are promising therapeutic tools in tomorrow's medicine, provided sufficient BsAb can be produced. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. Recombinant BsAb can be made by fusing single chain variable fragments (scFv) to a heterodimerization domain. We compare the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerization of BsAbs in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerized bispecific antibody was successful. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made by C-terminal fusion of scFv molecules to the L or Fd Fab chains. This gave rise to disulphide stabilized Fab-scFv BsAb (Bibody)or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerization of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerized format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the immunological functionality was demonstrated.     

Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector.

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.     

Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties

Escherichia coli (E. coli) is the most commonly used organism for expressing antibody fragments such as single chain antibody Fvs (scFvs). Previously, we have utilized E. coli to express well-folded scFvs for characterization and engineering purposes with the goal of using these engineered proteins as building blocks for generating IgG-like bispecific antibodies (BsAbs). In the study, described here, we observed a significant difference in the secondary structure of an scFv produced in E. coli and the same scFv expressed and secreted from chinese hamster ovary (CHO) cells as part of a BsAb. We devised a proteolytic procedure to separate the CHO-derived scFv from its antibody-fusion partner and compared its properties with those of the E. coli-derived scFv. In comparison to the CHO-derived scFv, the E. coli-derived scFv was found trapped in a misfolded, but monomeric state that was stable for months at 4 °C. The misfolded state bound antigen in a heterogeneous fashion that included non-specific binding, which made functional characterization challenging. This odd incidence of obtaining a misfolded scFv from bacteria suggests careful characterization of the folded properties of bacterially expressed scFvs is warranted if anomalous issues with antigen-binding or non-specificity occur during an engineering campaign. Additionally, our proteolytic methodology for obtaining significant levels of intact scFvs from highly expressed IgG-like antibody proteins serves as a robust method for producing scFvs in CHO without the use of designed cleavage motifs.     

Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity

Herpes simplex virus (HSV) recombinants are being developed as vaccine vectors for the expression of heterologous antigens. There is concern, however, that preexisting HSV immunity may decrease their effectiveness. We have addressed this issue in an animal model. Immunized mice were inoculated with a replication-defective HSV-1 vector that expressed the Escherichia coli beta-galactosidase protein as a model antigen. We assessed vector efficacy by analyzing the immunoglobulin G (IgG) antibody response and cellular proliferative response directed against beta-galactosidase. We report that the ability of the vector to induce antibody or proliferative responses was not diminished by preexisting immunity to HSV. Of further note, the anti-HSV and anti-beta-galactosidase IgG responses following vector administration were extremely durable in both immunized and naive mice. These results indicate that the ability of a replication-defective HSV-derived vaccine vector to elicit long-lived immune responses in mice is not impaired by prior HSV exposure.     

Production,purification and characterisation of genetically derived scFv and bifunctional antibody fragments capable of detecting illicit drug residues

We have generated a single chain antigen binding protein (scFv) recognising morphine. Variable regions of heavy (V(H)) and light (V(L)) chain antibody genes isolated from a murine immune repertoire were connected via a glycine-serine linker and cloned into the expression vector pAK 400. The scFv was produced in Escherichia coli JM83 yielding a functional protein of approximately M(r) 30000. Immunoaffinity chromatography using M3G-BSA-Sepharose column proved most effective for scFv purification. Purity was monitored by SDS-PAGE and Western blotting and the scFv characterised using ELISA and BIAcore. The scFv was capable of specifically binding free morphine in solution and was applicable to real sample analysis in saliva. In order to express a bivalent "minibody" the scFv gene was recloned into a vector containing a gene encoding a helix for dimerisation. The scFv was expressed as a protein of M(r) 75000 and retained its antibody binding capabilities. Cloning the scFv gene into a vector containing the bacterial alkaline phosphatase gene produced a bifunctional molecule, which retained the binding activity of the parental scFv along with the enzymatic activity of alkaline phosphatase.