Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

Adaptive laboratory evolution induced novel mutations in Zymomonas mobilis ATCC ZW658: a potential platform for co-utilization of glucose and xylose.

A systematic adaptive laboratory evolution strategy was employed to develop a potential Zymomonas mobilis strain with the ability to co-utilize glucose and xylose. Z. mobilis ATCC ZW658, a recombinant xylose fermenting strain, was subjected to adaptive laboratory evolution over a period of 200 days under strict selection pressure of increasing concentration of xylose. The evolved strain exhibited 1.65 times increase in the overall specific xylose utilization rate when compared with the parent strain. Furthermore, the strain displayed significantly improved performance in terms of co-fermentation of xylose in the presence of glucose with specific glucose and xylose utilization rate of 1.24 g g-1 h-1 and 1.34 g g-1 h-1, respectively. Altered phenotypic response of the evolved strain, in terms of improved xylose utilization, co-utilization of mixed sugars, enhanced growth, ethanol production, and reduced xylitol production has been explained by novel mutations, identified using next-generation sequencing, in xylose assimilating, metabolizing, and crucial regulatory pathway genes and key enzyme activity assays.

The µ1 72-96 loop controls conformational transitions during reovirus cell entry.

The reovirus outer capsid protein µ1 forms a lattice surrounding the viral core. In the native state, µ1 determines the environmental stability of the viral capsid. Additionally, during cell entry, µ1 undergoes structural rearrangements that facilitate delivery of the viral cores across the membrane. To determine how the capsid-stabilizing functions of µ1 impinge on the capacity of µ1 to undergo conformational changes required for cell entry, we characterized viruses with mutations engineered at charged residues within the µ1 loop formed by residues 72 to 96 (72-96 loop). This loop is proposed to stabilize the capsid by mediating interactions between neighboring µ1 trimers and between trimers and the core. We found that mutations at Glu89 (E89) within this loop produced viruses with compromised efficiency for completing their replication cycle. ISVPs of E89 mutants converted to ISVP*s more readily than those of wild-type viruses. The E89 mutants yielded revertants with second-site substitutions within regions that mediate interaction between µ1 trimers at a site distinct from the 72-96 loop. These viruses also contained changes in regions that control interactions within µ1 trimers. Viruses containing these second-site changes displayed restored plaque phenotypes and were capable of undergoing ISVP-to-ISVP* conversion in a regulated manner. These findings highlight regions of µ1 that stabilize the reovirus capsid and demonstrate that an enhanced propensity to form ISVP*s in an unregulated manner compromises viral fitness.

Cell entry-associated conformational changes in reovirus particles are controlled by host protease activity.

Membrane penetration by reovirus requires successive formation of two cell entry intermediates, infectious subvirion particles (ISVPs) and ISVP*s. In vitro incubation of reovirus virions with high concentration of chymotrypsin (CHT) results in partial digestion of the viral outer capsid to form ISVPs. When virions are instead digested with low concentrations of chymotrypsin, the outer capsid is completely proteolyzed to form cores. We investigated the basis for the inverse relationship between CHT activity and protease susceptibility of the reovirus outer capsid. We report that core formation following low-concentration CHT digestion proceeds via formation of particles that contain a protease-sensitive form of the µ1C protein, a characteristic of ISVP*s. In addition, we found that both biochemical features and viral genetic requirements for ISVP* formation and core formation following low-concentration CHT digestion are identical, suggesting that core formation proceeds via a particle resembling ISVP*s. Furthermore, we determined that intermediates generated following low-concentration CHT digestion are distinct from ISVPs and convert to ISVP*-like particles much more readily than ISVPs. These results suggest that the activity of host proteases used to generate ISVPs can influence the efficiency with which the next step in reovirus cell entry, namely, ISVP-to-ISVP* conversion, occurs.

Determinants of strain-specific differences in efficiency of reovirus entry.

Cell entry of reovirus requires a series of ordered steps, which include conformational changes in outer capsid protein µ1 and its autocleavage. The µ1N fragment released as a consequence of these events interacts with host cell membranes and mediates their disruption, leading to delivery of the viral core into the cytoplasm. The prototype reovirus strains T1L and T3D exhibit differences in the efficiency of autocleavage, in the propensity to undergo conformational changes required for membrane penetration, and in the capacity for penetrating host cell membranes. To better understand how polymorphic differences in µ1 influence reovirus entry events, we generated recombinant viruses that express chimeric T1L-T3D µ1 proteins and characterized them for the capacity to efficiently complete each step required for membrane penetration. Our studies revealed two important functions for the central δ region of µ1. First, we found that µ1 autocleavage is regulated by the N-terminal portion of δ, which forms an α-helical pedestal structure. Second, we observed that the C-terminal portion of δ, which forms a jelly-roll ß barrel structure, regulates membrane penetration by influencing the efficiency of ISVP* formation. Thus, our studies highlight the molecular basis for differences in the membrane penetration efficiency displayed by prototype reovirus strains and suggest that distinct portions of the reovirus δ domain influence different steps during entry.

Regulation of butanol biosynthesis in Clostridium acetobutylicum ATCC 824 under the influence of zinc supplementation and magnesium starvation.

Present study reports modulation in butanol biosynthesis in Clostridium acetobutylicum ATCC 824 under the influence of zinc supplementation or magnesium starvation either individually or in combination. An improvement in butanol titer from 11.83 g L-1 in control to 13.72 g L-1, 15.79 g L-1, and 19.18 g L-1 was achieved when organism was grown on magnesium starved, zinc supplemented and combined zinc supplemented-magnesium starved fermentation medium, respectively. The elevation in butanol biosynthesis was associated with raised glucose utilization, reduced ethanol production and early induction of solventogenesis. Change in these phenotypic traits of the organism may be attributed to multi-level modulation in central carbon metabolism e.g., upregulation of glycolytic pathway; upregulation in thiolase activity; key intermediate enzyme for biosynthesis of acids and solvent; upregulation in the activity of butyrylaldehyde dehydrogenase & butanol dehydrogenase, the enzymes responsible for butanol biosynthesis and downregulation in alcohol dehydrogenase, redirecting carbon flux from ethanol to butanol.

Dengue virus infection of SK Hep1 cells: inhibition of in vitro angiogenesis and altered cytomorphology by expressed viral envelope glycoprotein.

Dengue virus (DENV) infection of human endothelial cells has been implicated in the pathobiology of dengue hemorrhagic fever and dengue shock syndrome. However, the mechanisms by which DENV infections alter the functional physiology of endothelial cells remain incompletely understood. In the present study, we examined the susceptibility of a human liver sinusoidal endothelial cell line SK Hep1 to all four serotypes of DENV and studied the effect of the virus on in vitro angiogenesis. All four serotypes of DENV could infect the SK Hep1 cells, but showed variable cytopathic effects, the most pronounced being that of DENV-2. Electron microscopy of the infected cells showed significant ultrastructural changes. In vitro angiogenesis assays on DENV-2 exposed SK Hep1 cells in the matrigel system showed inhibition compared with the controls. Importantly, transfection and transient expression of the DENV-2 envelope glycoprotein (E) in these cells showed drastic alterations in cell shapes and the E protein could be localized by fluorescence microscopy in terminal knob-like structures. Therefore, SK Hep1, a human hepatic sinusoid-derived endothelial cell line, may constitute a potential model to study DENV-endothelial cell interactions in vitro, especially towards understanding the possible virus-induced changes in hepatic endothelium and its role in disease pathogenesis.