Annie-Paule Sibler

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

BackgroundIntrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.ResultsWe describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus.ConclusionThis new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA1 Arg, tRNA2 Arg, tRNAGly, tRNA2 Lys tRNALeu and tRNAPro are reported. Special structural features were found in tRNAPro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA2 Lys, which has a U excluded from basepairing and bulging out from the TΨC stem. The tRNA1 Arg, tRNA2 Lys and tRNALeu, which belong to two‐codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5‐carboxymethylaminomethyluridine. tRNAGly and tRNAPro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four‐codon family. However, tRNA2 Arg, which is a minor species and belongs to the CGN four‐codon family, has an unmodified A in the wobble position. This very unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA.

Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPV-positive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOH-terminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipid-mediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications. [Mol Cancer Ther 2007;16(5):1728–35]

In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgG1 Fab fragments

In vivo biotinylation of antibody fragments with a gene fusion approach is a realistic alternative to conventional in vitro chemical labelling. We have previously reported the construction of a vector system suitable for the bacterial expression of the binding fragment of antibody (Fab) genetically linked to the C-terminal domain of Escherichia Coli biotin carboxy carrier protein (BCCP*). A minor fraction of the expressed hybrids was biotinylated in vivo and therefore able to interact with streptavidin. We now show that the large majority of bacterially-expressed Fab-BCCP* fusions are labelled with biotin when plasmid-encoded biotin holoenzyme synthetase (BirA) is co-expressed. The yield of biotinylated Fab is maximal when overexpression of BirA is driven by a second compatible plasmid. We took advantage of this property to develop a novel filter assay for the rapid identification of recombinant Fab reacting with immunoglobulin. Starting with total RNA of two newly established murine hybridoma cell lines producing anti-human IgG1 antibodies, we selected in a single experiment the bacterial clones that expressed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated Fabs showed that they did not derive from a single B clone. In addition, we found that these recombinant Fabs labelled with biotin in vivo are useful for the specific detection of human IgG1 by a solid-phase immunoassay.

Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells.

Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions.

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.

The Primary Structure of tRNAs and Their Rare Nucleosides

Since the first determination of a primary structure of a tRNA by Holley et al. (1965), 113 structures have been published of which 108 are different. This number is sometimes difficult to calculate. Two structures are defined as different if they differ by one basic nucleotide, for example, if an A replaces a G, but not if they differ by posttranscriptional modifications. Some structures have been determined on mixtures of isoacceptors that were impossible to separate, like the one of beef liver tRNA Trp (Fournier et al. 1978). Figure 1 shows that this tRNA has several incompletely modified nucleotides, such as m 2 G in the amino acid stem, Cm in the anticodon loop, and m 7 G in the variable loop. But in two positions, C is partially replaced by D. If these Ds arise from a posttranscriptional modification of C, then we would have only two tRNAs Trp , one having a G in position 56 and the other having an A in this position. In fact, so far, only U → D heterogeneities have been found in tRNAs. There is no evidence that a C can be modified to a D. Therefore, we conclude that two more basic nucleotides are different in the tRNA Trp . There are several combinations possible between tRNAs differing in three different positions; we have at least three tRNAs Trp . In fact, by two-dimensional gel electrophoresis, five spots have now been found for this tRNA (M. Fournier, pers. comm.). It will be interesting to test if each functions as primer for reverse...

Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coliβ‐galactosidase conferred by the single‐chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short‐lived or destabilized intrabodies could affect the steady‐state level of target by redirecting it to the proteasomes. In the absence of antigen, the half‐life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with β‐galactosidase. Analysis of destabilized site‐directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of β‐galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST‐tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.

Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120.

The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.

Nucleotide sequence of a yeast mitochondrial threonine‐tRNA able to decode the C—U—N leucine codons

Sequencing studies of mitochondrial (mt) tRNAs and tRNA genes during the past two years led to the finding of unusual features in the codon reading patterns and in the genetic code in mitochondria [l-3]. The most striking example is that the U-G-A codon, instead of acting as a termination signal, is used to specify tryptophan in mitochondria [4,5]. Whereas this exception to the standard genetic code is found in mitochondria from various sources, there are others which differ from one organism to another [l-3], so there is not: even a common code in different mitochondria. In the yeast Saccharornyces cerevisiae, such an exception has been deduced from the observation that in the ATPase proteolipid amino acid sequence, the threonine residue at position 46 is encoded by the C-U-A codon for leucine [6,7]. This unexpected amino acid assignment for the C-U-A codon has been explained in [7] where the gene for a tRNATh bearing an unorthodox anticodon loop with a presumed anticodon U-A-C was sequenced. To determine whether this unique codon assignment is also found at the level of the corresponding functional threonine-tRNA and whether this tRNA is able to translate all the four codons of the leucine family C-U-N (N = A, G, U or C) into threonine, we have purified and sequenced the mt tRNAp.