Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume.

Bradyrhizobium is an endophytic bacterium under investigation as an efficient biofertilizer for sustainable legume-rice rotational cropping system. Monitoring and bio-imaging of this nitrogen fixing bacterium is essential for the study of plant-microbe evolution, soil microbiome, as well as quality control in organic farming. While phage display antibody technology has been widely used to generate recombinant antibody for myriad medical purposes, so far, this technology has been minimally applied in the agricultural sector. In this study, single-chain variable fragments (scFv) against two Bradyrhizobium strains SUTN9-2 (yiN92-1e10) and DOA9 (yiDOA9-162) were isolated from a human phage display antibody library. Specific binding of scFv was demonstrated by ELISA and confocal-immunofluorescence imaging techniques. Bradyrhizobium localization in both endophytic and bacteroid forms could be observed inside rice tissue and plant nodule, respectively. Moreover, successful application of the recombinant antibody for the evaluation of nodule occupancy was also demonstrated in comparison with standard GUS-staining method. The results of this study showed for the first time the potential use of human phage display scFv antibody for imaging and monitoring of Bradyrhizobium biofertilizer and thus could be further applied for point-of-detection of bacterial inoculum in the legume-rice rotational crop system. IMPORTANCE Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively. These two recombinant scFv antibodies could be used for precise detection of the rhizobia both in symbiosis with legume and endophyte in rice tissue by ELISA and immunofluorescent staining, during legume-rice rotational cropping system in the field. This methodology can be further employed for the study of other plant-microbe interactions and monitoring of biofertilizer in diverse sustainable cropping systems as well as in precision agriculture.

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results: : Increased efficiency was achieved by EnBase® compared to Luria­Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 µg/mL. Conclusion: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

Eight novel single chain antibody fragments recognising VP2 of foot-and-mouth disease virus serotypes A, O, and SAT 2.

Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic diversity among its serotypes, requiring several anti-FMDV antibodies for its laboratory diagnosis, which complicated the used techniques. To conquer this cumbersome, we developed a new panel of different single-chain fragment variable (scFv) for serotype-independent detection of FMDV. The recombinant VP2 capsid protein, as a relatively conserved protein among FMDV serotypes, was expressed in E. Coli, and injected in mice. Spleen's RNA was extracted for isolating the coding sequences of IgG variable domains that were assembled into repertoires of scFv. Phage library displaying scFv was constructed with ∼1.9 × 108 plaque forming units. Characterization of the library showed eight of unique scFvs, which were expressed as bacterial periplasmic proteins with apparent molecular weight of ∼27 kDa. Our data revealed the broad-spectrum binding affinity of the eight scFvs as both coating and tracing antibodies to FMDV serotypes A, O, and SAT 2.

Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy-chain antibody domain.

BACKGROUND: The variable domain of camelid heavy-chain antibodies (VHH) is increasingly being adapted to detect small molecules in various matrices. The insecticide carbaryl is widely used in agriculture while its residues have posed a threat to food safety and human health. RESULTS: VHHs specific for carbaryl were generated from an alpaca immunized with the hapten CBR1 coupled to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) based on the VHH C1 and the coating antigen CBR2-BSA was developed for the detection of carbaryl in cereals. This assay, using an optimized assay buffer (pH 6.5) containing 10% methanol and 0.8% NaCl, has a half-maximum signal inhibition concentration of 5.4 ng mL-1 and a limit of detection (LOD) of 0.3 ng mL-1 for carbaryl, and shows low cross reactivity (≤0.8%) with other tested carbamates. The LOD of carbaryl using the VHH-based ELISA was 36 ng g-1 in rice and maize and 72 ng g-1 in wheat. Recoveries of carbaryl in spiked rice, maize and wheat samples were in the range of 81-106%, 96-106% and 83-113%, respectively. Relative standard deviations of repeatability and intra-laboratory reproducibility were in the range of 0.8-9.2% and 2.9-9.7%, respectively. CONCLUSION: The VHH-based ELISA was highly effective in detecting carbaryl in cereal samples after simple sample extraction and dilution. © 2019 Society of Chemical Industry.

Development and identification of a fully human single-chain variable fragment 29 against TSLP.

Thymic stromal lymphopoietin is a key initiator for inducing Th2-type inflammation and a potential therapeutic target for allergic disease. In the present study, the naive human antibody library was enriched using human thymic stromal lymphopoietin (hTSLP) as an antigen by phage display. Single clones were randomly picked from the enriched antibody library after three rounds of selection, and these were expressed for enzyme-linked immunosorbent assay (ELISA). The positive single-chain fragment variables (scFvs) determined by ELISA were further identified by Western blot, Biacore, and flow cytometry. After three rounds of phage display, 35% of the scFv clones were positive by ELISA and could bind well with hTSLP. Further identification revealed that scFv29 had satisfactory characteristics. The scFv29 was specific to hTSLP, and had no cross-reaction with hIL-33, hIL-4, and hIL-13. The scFv29 could bind to hTSLP in competition with the TSLP receptor and could also bind to mouse TSLP. Cellular experiments revealed that mTSLP could stimulate myeloid dendritic cell (DC) to mature, and scFv29 blocking could reduce the maturation rate of DC. These findings suggest that scFv29 could be used as a neutralizing antibody to block the signaling of TSLP, and this work provides the foundation for further study of the therapeutic roles of TSLP in allergic inflammation diseases.

RAGE-specific single chain Fv for PET imaging of pancreatic cancer.

Noninvasive detection of both early pancreatic neoplasia and metastases could enhance strategies to improve patient survival in this disease that is notorious for an extremely poor prognosis. There are almost no identifiable targets for non-invasive diagnosis by positron emission tomography (PET) for patients with pancreatic ductal adenocarcinoma (PDAC). Over-expression of the receptor for advanced glycation end products (RAGE) is found on the cell surface of both pre-neoplastic lesions and invasive PDAC. Here, a RAGE-specific single chain (scFv) was developed, specific for PET imaging in syngeneic mouse models of PDAC. An anti-RAGE scFv conjugated with a sulfo-Cy5 fluorescence molecule showed high affinity and selectivity for RAGE expressing pancreatic tumor cells and genetically engineered KRASG12D mouse models of PDAC. An in vivo biodistribution study was performed with the 64Cu-radiolabled scFv in a syngeneic murine pancreatic cancer model, demonstrating both the feasibility and potential of an anti-RAGE scFv for detection of PDAC. These studies hold great promise for translation into the clinic.

Survival of Escherichia coli harboring nucleic acid-hydrolyzing 3D8 scFv during RNA virus infection.

Previously, Escherichia coli harboring the codon-optimized 3D8scFv gene (E. coli 3D8scFv) was developed as a feed additive for use in preventing norovirus infection. Here, we evaluated whether the 3D8scFv gene affects the colonization of E coli when E. coli 3D8scFv passes through the mouse gastrointestinal tract. To determine the colonization ability of E. coli 3D8scFv, E. coli cells with or without the 3D8scFv gene were fed to mice. Total DNA was extracted from the animals' stools, stomach, small intestine and colon. All samples were amplified using 3D8scFv gene-specific primer sets. E. coli 3D8scFv begins to be excreted 1 h after feeding and that all E. coli 3D8scFv cells were excreted between 12 and 24 h after the last feeding of the cells. The previously measured gastrointestinal transit time of the mice was between 8 h and 22 h. The results of this study therefore show that E. coli 3D8scFv cannot colonize the gastrointestinal tracts of mice. In addition, if the purified 3D8 scFv protein is used as a feed additive, any associated E. coli 3D8scFv bacteria will not colonize the gastrointestinal tracts of the livestock. Thus, this feed additive meets the safety assessment criteria for the commercial use of bacteria.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged ß2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Detection and purification of backbone-cyclized proteins using a bacterially expressed anti-myc-tag single chain antibody.

A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb. However, the co-expression of a chaperone, Skp, was absolutely necessary for the activity even when the protein was expressed in a soluble manner. We could purify about 4 mg of 9E10 scAb from 1 l of culture, and the resulting scAb could be used to detect and purify the backbone-cyclized protein as the parental full-length 9E10. Moreover, the immunoaffinity resin prepared using 9E10 scAb could be regenerated several times after the elution of bound proteins using an acid, which added more value to the ready preparation of the active antibody in bacteria.

Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies.

In the present study, a Cry1F-immunized rabbit phage display library (6.96 × 108 cfu/mL) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFv's were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed in Escherichia coli HB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for Cry1F toxin. It indicated the 50% inhibition of the control (IC50) was 11.56 ng/mL and the detection limit (IC10) was 0.18 ng/mL and showed weak cross-reactivities for Cry1Ab (2.8%), Cry1Ac (1.3%), and Cry1B, Cry1C, Cry1Ie, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2-99.6%, and the coefficients of variation were in the range of 2.5-10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.

Improved Soluble ScFv ELISA Screening Approach for Antibody Discovery Using Phage Display Technology.

Phage display technology (PDT) is a powerful tool for the isolation of recombinant antibody (Ab) fragments. Using PDT, target molecule-specific phage-Ab clones are enriched through the "biopanning" process. The individual specific binders are screened by the monoclonal scFv enzyme-linked immunosorbent assay (ELISA) that may associate with inevitable false-negative results. Thus, in this study, three strategies were investigated for optimization of the scFvs screening using Tomlinson I and J libraries, including (1) optimizing the expression of functional scFvs, (2) improving the sensitivity of ELISA, and (3) preparing different samples containing scFvs. The expression of all scFv Abs was significantly enhanced when scFv clones were cultivated in the terrific broth (TB) medium at the optimum temperature of 30 °C. The protein A-conjugated with horseradish peroxidase (HRP) was found to be a well-suited reagent for the detection of Ag-bound scFvs in comparison with either anti-c-myc Ab or the mixing procedure. Based on our findings, it seems there is no universal media supplement for an improved expression of all scFvs derived from both Tomlinson I and J libraries. We thus propose that expression of scFv fragments in a microplate scale is largely dependent on a variety of parameters, in particular the scFv clones and relevant sequences.

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains.

BACKGROUND: Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv. RESULTS: Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L-1) and highest space-time-yield (8 mg L-1 h-1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac). CONCLUSIONS: The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space-time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.

Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv.

A Combinatory Antibody-Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries.

We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.

[Construction of anti-B7-H4-scFv library and screening and identification of anti-B7-H4-scFv].

Objective To construct the ribosome display library of anti-B7-H4 extracellular domain, and select the antibody with high specificity. Methods The cDNA of B7-H4 extracellular domain was amplified from A549 cells by reverse transcription PCR (RT-PCR). To express ectodomains of B7-H4, the sequence of B7-H4 gene, which encodes the B7-H4 extracellular domains, was inserted into plasmid pET-28a(+). The purified recombinant protein of B7-H4 extracellular domain was used to immunize BALB/c mice. The total RNA was extracted from the spleen of BALB/c mice which had been immunized with B7-H4 recombinant protein. The genes of VH, Vκ and VH/Vκ were amplified separately by RT-PCR and splicing by overlap extension PCR (SOE-PCR). The gene of VH/ Vκ was ligated into pUM19-T vector and the ligated sample was transformed into competent E.coli DH5α. The resulting plasmid was isolated and then subjected to sequencing to verify the gene sequence. TNT(R)T7 Quick for PCR DNA kit was used to translate and screen the anti-B7-H4-scFv in vitro from the ribosome display library. Western blotting and an indirect ELISA were performed to detect the specificity of anti-B7-H4-scFv. Results The right sequences of VH, Vκ and VH/Vκ were acquired, which were 439, 680 and 1098 bp in length, respectively. The analysis of specificity demonstrated that the anti-B7-H4-scFv screened from the ribosome display library had a high specific combining ability with B7-H4. Conclusion The experiment has successively constructed the ribosome display library of anti-B7-H4 extracellular domain, and selected the anti-B7-H4-scFv which has a high specific binding ability with recombinant protein of B7-H4 extracelluar domain.

A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation.

Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.

Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.

Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (V(H )and V(L)) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS-PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the V(H) and V(L) chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection.

Single Chain Antibodies as Tools to Study transforming growth factor-ß-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-ß signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-ß signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-ß signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.