In (B), the canonical NLS directs scFv-D5 intrabody into nuclei (asterisks), whereas the inverted (rNLS) sequence is nonfunctional (arrows indicate cytoplasmic localization pattern)

In (B), the canonical NLS directs scFv-D5 intrabody into nuclei (asterisks), whereas the inverted (rNLS) sequence is nonfunctional (arrows indicate cytoplasmic localization pattern). is usually improved by an overall unfavorable charge at cytoplasmic pH and reduced hydrophilicity. We hypothesize that ionic repulsion and poor hydrophobic interactions compensate, to different extents, for impaired disulfide bond formation in cytoplasm, thereby decreasing the risk for intrabody aggregation. As proof of theory, we demonstrate that this soluble expression of an aggregation-prone positively charged intrabody is usually modestly enhanced via or acidification using highly charged peptide tags X-376 (3XFLAG tag, SV40 NLS). These findings suggest that simple sequence analysis and electrostatic manipulation may aid in predicting and engineering solubility-enhanced intrabodies from antibody libraries for intracellular use. orientation: Kozak sequence-start-intrabody-HA-(Gly4Ser)4-EGFP-stop. Amino-acid changes in recoded C4 intrabody (rcC4) were launched by re-synthesizing cDNA with the desired changes (GeneArt). The HA tag on scFv-D5 was replaced via re-amplification of intrabody cDNA using a reverse primer that launched a 3XFLAG epitope tag [(DYKDDDK)3], followed by re-ligation upstream of X-376 (Gly4Ser)4-EGFP in pcDNA3.1(?) X-376 at identical restriction sites. Similarly, canonical (TPPKKKRKV) or inverted (VKRKKKPPT) SV40 nuclear localization sequences were cloned onto the 3 end of scFv-D5 and rcC4 via re-amplification of HA-tagged intrabody cDNA using corresponding reverse primers that launched these sequences, and then re-ligated upstream of (Gly4Ser)4-EGFP in pcDNA3.1(?) at identical restriction sites. Expression vectors for nuclear localization transmission (NLS)-mRFP, a live-cell fluorescent nuclear marker, httex1-25Q-mRFP and httex1-72Q-EGFP were explained previously (Kvam (Ewert stability according to the best predictive and structural modeling methods available at the time (Ewert for improved stability and folding, exhibits reduced functional efficacy in blocking the intracellular aggregation of X-376 mutant huntingtin protein compared with the original scFv-C4 intrabody. Huntingtin aggregates were scored in ST14A striatal progenitor cells after transient co-transfection of httex1-72Q-GFP with rcC4, scFv-C4 or empty vector, as explained in the Materials and methods section. Statistical significance from vector control (***< 0.0001) was determined by ANOVA (= 4). (B) Fluorescently labeled rcC4 forms aggregates in cell cytoplasm and is detected at reduced detergent-soluble levels in the constant state compared with the original scFv-C4 intrabody. Cells expressing rcC4-HA-EGFP or C4-HA-EGFP in the absence of antigen were scored for fluorescent intrabody aggregates among three impartial replicates, as explained in the Materials and methods section. Soluble (inset i) and detergent-insoluble (inset ii) intrabody fractions were analyzed by western blot (non, non-transfected cells). Estimates of intrabody net charge and GRAVY were inferred from amino-acid sequence data. To investigate the basis for this phenomenon using current methods, we calculated the net charge and hydropathicity of rcC4 using amino-acid sequence data (Supplementary Fig. S2). Unlike scFv-C4, which is usually soluble in cell cytoplasm (Table?II; net charge ?0.5, GRAVY score ?0.282), rcC4 is strikingly basic (net charge +1.5) and more hydrophilic (GRAVY score ?0.303). These findings suggested that rcC4 may in fact be aggregation-prone in the cytoplasmic environment, despite extensive engineering to improve its stability. Indeed, live-cell imaging revealed that GFP-labeled rcC4 intrabody is usually significantly aggregation-prone in cell cytoplasm (Fig.?2B) and was consequently detected at reduced detergent-soluble levels in the constant state compared with scFv-C4 intrabody (Fig.?2B, inset). Importantly, we observed that non-aggregated rcC4 intrabody sequestered a native huntingtin reporter protein (httex1-25Q) as efficiently as our initial scFv-C4 intrabody using a classical antibody-antigen re-targeting assay for detecting intracellular proteinCprotein interactions (Sibler for specific intracellular use. Influence of net charge and hydropathicity around the soluble expression of camelid VHH intrabodies We next tested whether protein net charge and hydropathicity also influence the soluble expression of camelid VHH intrabodies in mammalian cell cytoplasm. Camelid VHHs are experimentally attractive single-domain antibody fragments because they have evolved to fold properly in the absence of light chains and are therefore considered to be innately more stable than corresponding human single-domain VHs (Davies and Riechmann, 1996; Muyldermans, 2001). Indeed, intracellular experiments show that camelid VHHs are readily flexible as soluble Rabbit polyclonal to IL1B cytoplasmic intrabodies (Rothbauer neurotoxin light-chain protease domains (BoNT LCs; C.B. Shoemaker, manuscript in preparation). We fused three BoNT LC serotype A-binding VHHs and two BoNT LC serotype B-binding VHHs (Table?III) to GFP (Fig.?1A) in order to create VHH-GFP chromobodies (Rothbauer or acidification improves soluble expression of an aggregation-prone, positively charged human scFv intrabody As a proof of theory for X-376 the observed relationship between net charge and intrabody solubility, we next tested whether electrostatic manipulation of an aggregation-prone, positively charged intrabody (scFv-D5) selected from your human scFv Tomlinson library (Fig.?1B) can reduce aggregation and improve soluble intrabody expression in mammalian cell cytoplasm. Toward this goal, we replaced the existing HA epitope tag on scFv-D5 (Fig.?1A) with.