ISWI chromatin remodelers mobilize nucleosomes to control DNA accessibility. Complexes isolated to date pair one of six regulatory subunits with one of two highly similar ATPases. However, we find that each endogenously expressed ATPase co-purifies with every regulatory subunit, substantially increasing the diversity of ISWI complexes, and we additionally identify BAZ2B as a novel, seventh regulatory subunit. Through reconstitution of catalytically active human ISWI complexes, we demonstrate that the new interactions described here are stable and direct. Finally, we profile the nucleosome remodeling functions of the now expanded family of ISWI chromatin remodelers. By revealing the combinatorial nature of ISWI complexes, we provide a basis for better understanding ISWI function in normal settings and disease.

One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-positive individuals after exposure to an infectious reagent or disease etiology followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and only represent a small percentage of the total B cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, we demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required, without the need for sequencing of genetic material. Peripheral pAbs from a CMV exposed individual were purified by glycoprotein B antigen-affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufactured in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, we found that these mAbs accounted for 1–2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.

BackgroundProtein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies. In this approach, a signal peptide is fused to the N-terminus of the heterologous protein. The signal peptide mediates translocation of the heterologous protein from the cytoplasm to the periplasm and is cleaved during the translocation process. It was previously shown that optimization of the translation initiation region (TIR) which overlaps with the nucleotide sequence of the signal sequence improves the production of heterologous proteins. Despite the progress, there is still room to improve yields using secretion as a means to produce protein complexes such as full-length monoclonal antibodies (mAbs).ResultsIn this study we identified the inefficient secretion of heavy chain as the limitation for full-length mAb accumulation in the periplasm. To improve heavy chain secretion we investigated the effects of various signal peptides at controlled TIR strengths. The signal peptide of disulfide oxidoreductase (DsbA) mediated more efficient secretion of heavy chain than the other signal peptides tested. Mutagenesis studies demonstrated that at controlled translational levels, hydrophobicity of the hydrophobic core (H-region) of the signal peptide is a critical factor for heavy chain secretion and full-length mAb accumulation in the periplasm. Increasing the hydrophobicity of a signal peptide enhanced heavy chain secretion and periplasmic levels of assembled full-length mAbs, while decreasing the hydrophobicity had the opposite effect.ConclusionsThis study demonstrates that under similar translational strengths, the hydrophobicity of the signal peptide plays an important role in heavy chain secretion. Increasing the hydrophobicity of the H-region and controlling TIR strengths can serve as an approach to improve heavy chain secretion and full-length mAb production in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0445-3) contains supplementary material, which is available to authorized users.

Bacillus spp. can exert plant growth-promoting effects and biocontrol effects after effective colonization, and bacterial chemotaxis toward plant root exudates is the initial step to colonize. Under biotic stress, plants are able to alter their root exudates in order to attract or avoid different types of microbes. Hence, Bacillus chemotaxis toward root exudates after pathogen infection is crucial for exerting their beneficial effects. In this study, B. amyloliquefaciens OR2-30 strain, which exhibited greater chemotaxis ability toward maize root exudates after Fusarium graminearum infection, was screened from 156 rhizosphere microorganisms. The infected maize root exudates were further confirmed to improve the swarming and biofilm formation ability of OR2-30 strain. Chemotaxis, swarming and biofilm formation ability were able to influence bacterial colonization. Indeed, the OR2-30 strain displayed more effective colonization ability in maize rhizosphere after F. graminearum inoculation. Moreover, lipopeptides produced by OR2-30 were identified as iturins and responsible for suppressing F. graminearum growth. Further study showed that lipopeptides suppressed the growth of F. graminearum by inhibiting conidia formation and germination, inducing reactive oxygen species (ROS) production and causing cell death in mycelium. Eventually, OR2-30 strain increased maize resistance against F. graminearum. These results suggested that maize root exudates could recruit B. amyloliquefacines OR2-30 after F. graminearum infection, OR2-30 then suppress the F. graminearum by producing lipopeptides, such as iturins, to protect maize.

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.