Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.

Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7±2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

Formation of functional, non-amyloidogenic fibres by recombinantBacillus subtilisTasA

Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In theBacillus subtilisbiofilm matrix the extracellular fibres of TasA are essential. Here a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted fromB. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid-like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enableB. subtilisto remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross-complement theB. subtilis tasAdeletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.ContributionsConceived and designed the experiments: CE, EE, RG, CEM, RJM, MS, NSW; Performed the experiments: KB, LC, CE, EE, PKF, RG, CEM, RJM, MS, TS; Contributed new analytical tools: CE, EE, RG, TS; Analysed the data: CE, EE, CEM, RJM, MS, LCS, NSW; Wrote the paper: EE, RJM, CEM, MS, NSW.

DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4 MDa motor complex that traffics organelles, vesicles and macromolecules towards microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterise 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor BICD2. Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2, and are therefore expected to result in linkage of cargoes to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

Metabolomic analysis of riboswitch containing E. coli recombinant expression system

In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein inEscherichia colicells.

RECOMBINANT THERMOTOLERANT PHYTASE PRODUCED IN E.COLI

Phytic acid (myo-inositol hexakisphosphate) and its salts (phytates) are the major storage form of phosphorus in plants. Monogastric animals including hogs, poultry, and fish cannot utilize phytates as a source of phosphorus unless they are enzymatically destroyed with exogenous enzyme—phytase. Phytases are added to fodder in ever increasing dosage to improve utilization of plant-derived phosphorus because this reduces dependence of farms on inorganic fodder phosphates. Because of technological considerations, feed phytases have to withstand elevated temperatures (60-80°C), which are used during preparation of fodder. Enzymatic feed additives are becominutesg of high demand in Kazakhstan, and development of domestic technologies for production of agricultural enzymes is an ongoing challenge to the country’s biotechnology.Objectives: To develop a system for recombinant expression of industrially important thermotolerantphytase and confirm activity and thermal stability of the recombinantly expressed enzyme.Methods: De novo gene synthesis, expression of 6xHis-tagged protein in E.coli, immobilized metal affinity chouromatography, biochemical tests for activities of phosphatase and phytase.Results: Thermotolerantphytase was produced in E.coli using recombinant expression system. The obtained enzyme had phosphatise activity (hydrolyzed p-nitrophenyl phosphate) and phytase activity (hydrolyzed sodium phytate). The recombinant phytase tolerated increase of incubation temperature up to 70°C and demonstrated increase in activity towards phytate with increase in the reaction temperature in the range 30°C-70°C.Conclusion: Described gene and expression system have prospects of utilization in development of pilot industrial production of phytase in the country.

New Efficient Recombinant Expression System To Engineer Candida antarctica Lipase B

ABSTRACT Here, we report the use of Yarrowia lipolytica as a versatile expression host for developing protein engineering approaches to modify the properties of Candida antarctica lipase B. A reliable screening protocol was defined and validated using a saturation mutagenesis library, yielding mutants displaying higher catalytic efficiencies than the wild-type enzyme.

Structural Characterization of the 1918 Influenza Virus H1N1 Neuraminidase

ABSTRACT Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 “Spanish flu” (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-Å and 1.45-Å resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.