Expression Of Antibody Fragments Research Articles

Cloning and expression of antibody fragment (Fab) II: Effect of expression hosts on light and heavy chain gene expression

Protein expression stoichiometry of individual subunits plays a critical role in the formation of multi-domain functional therapeutic proteins like antibody fragments. In this study, we have evaluated light to heavy chain ratio (LC: HC) of rHu Ranibizumab expressed using duet expression vector in six E. coli strains. The LC: HC ratio at the protein level was determined using RP-HPLC whereas transcript abundance of LC and HC at the mRNA level was determined using RTqPCR. Protein level study as well as the transcript abundance profiles of the LC and HC genes were observed to be differentially expressed across the E. coli strains. BL21 STAR (DE3) and Origami 2 strains resulted in a nearly equal expression ratio of LC and HC genes. Untargeted shotgun proteomics analysis was performed to identify the differentially expressed proteins. Total 1083 proteins were identified in the spectral IDA library, while 244 and 149 proteins were differentially expressed in Origami 2 and BL21 STAR (DE3) respectively. These proteins are associated with translation and nucleotide biosynthesis pathways. Obtained results provide insights into the impact of different E.coli host strains on LC: HC ratio, the probable reasons behind the unbalanced gene ratio, and provide useful transcriptomic and proteomics analysis protocols.

Design of a novel switchable antibody display system in Pichiapastoris.

Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomycescerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichiapastoris (Komagataellaphaffii). Establishment of surface display in P.pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments(DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P.pastoris formaldehyde dehydrogenase (FLD1) promoter, S.cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyceslactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S.cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. KEY POINTS: • First report of antibody display in P.pastoris using episomal plasmids. • Identification of genetic elements conferring highest levels of antibody display. • Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.

Cloning and expression of antibody fragment (Fab) I: Effect of expression construct and induction strategies on light and heavy chain gene expression

Dual promoter expression constructs offer time and cost-effective alternatives to produce multi-domain proteins like antibody fragments. This investigation is focused on understanding the effect of expression construct (dual promoter vs. co-transformation strategy), codon optimization, and induction strategies on yield and expression stoichiometry of LC and HC genes of antibody fragment at shake-flask and bioreactor scale. rHu biosimilar Ranibizumab was selected as a model protein for the study. Expression stoichiometry of HC and LC gene at mRNA level was studied using RTqPCR, whereas protein expression level was studied quantitatively using RP-HPLC and SDS-PAGE analysis. In the case of dual promoter expression construct, it was observed that LC gene cloned in the MCS1 of the duet vectors has > 2-fold expression than the HC gene, cloned in the MCS2. Transcript abundance profile of the HC and LC genes determined at different time intervals post-induction shows a difference in the gene expression at the transcriptional level. Comparative analysis of dual promoter and co-transformation strategy shows better stoichiometry in co-transformation (1:1.3), whereas higher protein yield in a dual expression system (>2.4 fold). The use of lactose and galactose as inducers show higher Fab yield of 2.30 ± 0.03 g/L and 2.81 ± 0.06 g/L with expression stoichiometry of 1:1.9 and 1:2 (HC: LC) respectively than IPTG-based induction with a protein yield of 1.40 ± 0.02 g/L.

A novel strategy for efficient expression of an antibody fragment in Escherichia coli: ranibizumab as a case study

AbstractBACKGROUNDComplex biotherapeutics such as non‐glycosylated antibody fragments can be easily manufactured in Escherichia coli due to advancements in recombinant DNA technology. Ranibizumab is one such example of a complex, non‐glycosylated protein used to treat age‐related macular degeneration and macular oedema. Ranibizumab, a humanized monoclonal antibody fragment, is often expressed as inclusion bodies using the E. coli expression system. In this study, we propose a novel strategy for achieving efficient expression of antibody fragments in E. coli BL21 (DE3). Here, the whole antibody fragment (heavy chain + light chain of ranibizumab) has been engineered and cloned in a pET series vector under single promoter belonging to a prokaryotic host along with an extra ribosome binding site to allow equal expression of the light and heavy chains.RESULTSWe demonstrate heterologous expression of ranibizumab with a protein concentration of 0.4 ± 0.019 mg mL–1, confirmed using reversed‐phase high‐performance liquid chromatography with the double copy clone. The reduced and refolded antibody fragment have been analytically characterized using liquid chromatography–mass spectrometry, where masses of the in‐house clone were in correspondence with marketed product. Native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR) analyses were performed to confirm the formation of purified and active recombinant product. Binding kinetics to the target analyte vascular endothelial growth factor of refolded in‐house product was found to be similar to the marketed product as per SPR (12.7 nmol L−1 vs. 10.4 nmol L−1).CONCLUSIONCloning and process optimization have been performed to enhance expression yield. The proposed strategy offers an efficient approach for enhanced production of antibody fragments in microbial hosts. © 2021 Society of Chemical Industry (SCI).

Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion

Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of post­translational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from invitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E.coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.

RSM-based Model to Predict Optimum Fermentation Conditions for Soluble Expression of the Antibody Fragment Derived from 4D5MOC-B Humanized Mab in SHuffle™ T7 E. coli.

Overexpression of the EpCAM in epithelial-derived neoplasms makes this receptor a promising target in antibody-based therapy. Due to the lack of N-glycosylation, Escherichia coli (E. coli) seems to be the most appropriate choice for the expression of antibody fragments. However, developing a robust and cost-effective process that produces consistent therapeutic proteins from inclusion bodies is a major challenge. Undoubtedly, it can be circumvented by the soluble expression of these proteins. Utilization of numerous genetically modified hosts and optimization of cultivation conditions are two effective approaches widely used to overcome the insolubility problem. Due to the cytoplasmic expression of DsbC and the ability to the correct formation of disulfide bonds, the Shuffle™ T7 strain can be a suitable host for the soluble production of recombinant proteins. Here, Box-Behnken design (BBD)- Response surface methodology (RSM) modeling was employed to develop optimized culture conditions for 4D5MOC-B scFv fragment production in SHuffle™ T7 strain while solubility and production level were considered as responses. Although both responses were significantly influenced by post-induction temperature, cell density at induction time, and IPTG concentration, the temperature had the largest effect. The maximum experimental soluble protein obtained by adding 1 mM of IPTG into the M9 medium when the cell density reached 0.7 at 23 ᵒC was 693.56 µg/mL which was in good correlation with the predicted value of 720.742 µg/mL. Predictable total expression value was also experimentally verified. This strategy can be scaled-up for the production of large amounts of scFvs from SHuffle™ T7 E. coli to facilitate their potential applications as therapeutic and diagnostic agents.

Expressing of Recombinant VEGFR2-specific Nanobody in Baculovirus Expression System.

Background: Baculovirus expression system, introduced more than 20 years ago, is considered as a useful tool for large and complex eukaryotic recombinant protein production. A baculovirus expression vector is a recombinant virus which desired foreign protein coding sequences is under control of the virus gene promoter. Baculovirus only infects insect cells and do not normally infect vertebrates therefore, they possess no risk of biological risks for human.Objectives: The aim of this study was to recombinant expression of vascular endothelial growth factor (VEGF) reseptor-2 specific Nanobody in the baculovirus expression systemMaterials and Methods: Gene of specific Nanobody against the VEGF reseptor-2 that called 3VGR19 was cloned and expressed in baculovirus system. Results: 3VGR19 Nanobody gene was amplified by Polymerase Chain Reaction (PCR) using the specific primers, and was cloned in pFastBac HTA plasmid. DH10Bac bacteria was transformed with resulted donor plasmid. The cultured Sf9 insect cell line was transfected with recombinant bacmid, and finally, the expression and purification of 3VGR19 was confirmed in insect cells. Conclusions: In conclusion, Transient infection of insect cells with baculovirus can be a promising technology for expression of antibody fragments.

Production of a Recombinant Single-Domain Antibody for Gluten Detection in Foods Using the Pichia pastoris Expression System.

The detection of gluten in foodstuffs has become a growing concern in food allergen management as a result of the high ratio of population sensitive to the main gluten-containing cereals. In this study, a promising single-domain antibody previously isolated by phage display (dAb8E) was produced in Pichia pastoris resulting in high levels of the antibody fragment expression (330 mg/L). The purified dAb8E was proved to specifically bind to gluten proteins from wheat, barley and rye, exhibiting no cross reaction to other heterologous species. The dynamic range of the sandwich enzyme-linked immunosorbent assay (ELISA) covered 0.1 to 10 µg/mL of gliadin, reaching a limit of detection of 0.12 µg/mL. When experimental binary mixtures of the target cereals were analyzed, the limit of detection was 0.13 mg/g, which would theoretically correspond to gluten concentrations of approximately 13 mg/kg. Finally, thirty commercially available food products were analyzed by means of the developed assay to further confirm the applicability of the dAb8E for gluten determination. The proposed methodology enabled the generation of a new gluten-specific nanobody which could be used to guarantee the appropriate labelling of gluten-free foods.

Development of a time-resolved fluoroimmunoassay for measuring plasma growth hormone in Nile tilapia (Oreochromis niloticus)

Growth hormone is a hormone secreted from the pituitary and is involved in the regulation of most major physiological processes such as growth, development and metabolism. Therefore, an accurate and sensitive detection method is needed for the detection of tilapia serum Gh level. Phage display technology is widely used in the expression of antibody fragments, in which fragments of antibodies are expressed as a fusion with phage proteins and are displayed on the phage surface for easy screening. Time-resolved fluorescence immunoassay (TRFIA) is a microanalysis method developed nearly two decades ago and is one of the most sensitive analytical techniques. With the use of a special lanthanide, the detection background can be distinguished, which can greatly improve the sensitivity of detection.In this report, we cloned the VH and VL DNA fragments from the lymphocytes of rabbits immunized with recombinant Gh and assembled them with a linker to form a single-chain variable fragment (scFv) gene pool. Using phage display technology, we isolated scFv DNA fragments from the pool, which encode a protein that specifically binds to tilapia Gh. We then established Eu-DTTA-based TRFIA for measuring plasma Gh in tilapia. The sensitivity of double antibody sandwich Gh-TRFIA was 0.225 ng/ml, and the linear range of the standard curve was 0.225–250 ng/ml. The intra- and interassay coefficients of variation (CVs) were <9.1 and <4.5%, respectively. The cross-reactivities (CRs) of 1 μg/ml recombinant tilapia somatolactin (rtSl), prolactin (rtPrl) and thyroid-stimulating hormone beta subunit (rtTshb) were 0.042%, 0.472% and 0.036%, respectively. The sensitivity of direct competitive Gh-TRFIA was 0.208 ng/ml, and the linear range of the standard curve was 0.208–500 ng/ml. The intra- and interassay CVs were <4.8 and <7.1%, respectively. The CRs of 1 μg/ml rtSl, rtPrl and rtTshb were 0.041%, 0.079% and 0.073%, respectively.In conclusion, Gh-TRFIA is a safe (no concerns about radioactive isotopes), economical, and efficient detection method for the quantification of plasma Gh. Thus, the application of phage display technology for antibody screening and the use of TRFIA for tilapia Gh detection are conducive to research in the field of fish endocrinology.

Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host.

In this review, we summarize the current knowledge concerning the eukaryotic protozoan parasite Leishmania tarentolae, with a main focus on its potential for biotechnological applications. We will also discuss the genus, subgenus, and species-level classification of this parasite, its life cycle and geographical distribution, and similarities and differences to human-pathogenic species, as these aspects are relevant for the evaluation of biosafety aspects of L. tarentolae as host for recombinant DNA/protein applications. Studies indicate that strain LEM-125 but not strain TARII/UC of L. tarentolae might also be capable of infecting mammals, at least transiently. This could raise the question of whether the current biosafety level of this strain should be reevaluated. In addition, we will summarize the current state of biotechnological research involving L. tarentolae and explain why this eukaryotic parasite is an advantageous and promising human recombinant protein expression host. This summary includes overall biotechnological applications, insights into its protein expression machinery (especially on glycoprotein and antibody fragment expression), available expression vectors, cell culture conditions, and its potential as an immunotherapy agent for human leishmaniasis treatment. Furthermore, we will highlight useful online tools and, finally, discuss possible future applications such as the humanization of the glycosylation profile of L. tarentolae or the expression of mammalian recombinant proteins in amastigote-like cells of this species or in amastigotes of avirulent human-pathogenic Leishmania species.

Single-step colony assay with autoinduction of scFv expression for the screening of antibody libraries.

We present a simple colony assay method for screening antibody libraries based on autoinduction of antibody fragment expression. This protocol eliminates the need for colony size monitoring and a separate induction step for single-chain Fv (scFv) antibody fragment expression. Here, scFvs are expressed in an automatic and timely fashion during the assay, resulting in high yields of positive clones and substantial time savings. The method was used successfully to establish monoclonal scFvs with high affinity and specificity against human IgG.

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse.

Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 1012 vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH in vivo. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram.

The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to &lt;em&gt;S&lt;/em&gt;-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

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.

Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.

BackgroundDisulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations.ResultsHere we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active.ConclusionsOur results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0419-5) contains supplementary material, which is available to authorized users.

Efficient Expression of Antibody Fragments with the Brevibacillus Expression System

Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for production in bacteria. They also are useful in applications requiring intracellular delivery and for further engineering toward molecules possessing multiple custom functions. An expression system based on Brevibacillus is characterized by high efficiency and simple genetic recombination for secretory production. The Brevibacillus expression system has been successfully utilized for the efficient production of antibody fragments, e.g., scFvs (single-chain antibody fragments) comprising heavy-chain and light-chain variable domains, linked by a spacer sequence. Expression in fusion with a Halobacterium-derived secretory protein was shown to confer enhanced productivity. In the case of Fabs, productivity as high as 100 mg/L was accomplished in a simple system, i.e., shake flask cultures. The Brevibacillus expression system offers several advantages, shared by other bacterial systems, such as E. coli, in particular, for the ease in genetic engineering and culture production.

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells.

Production of single chain fragment variable (scFv) antibodies in Escherichia coli using the LEX™ bioreactor

For proteome research, antibodies against a growing number of antigens must be generated and characterized. The high throughput generation of antibody fragments, using in vitro selection, requires bacterial expression of antibody fragments. This created a need to establish an expression method to improve the parallel production of many antibody fragments. In this study, we describe the development of a high throughput bacterial production method for single chain fragment variables (scFvs) using shaking flasks or the LEX™ bioreactor. We compared the influence of a set of production parameters on Escherichia coli production of four different scFv. The results led to an optimized protocol for the parallel production of multiple antibody fragments.